The US has imposed sanctions on seven Russian individuals and 17 companies it says are linked to President Vladimir Putin's "inner circle".
The White House said the move was a response to "Russia's continued illegal intervention in Ukraine".
Those targeted include Igor Sechin, head of oil giant Rosneft, and Sergei Chemezov of the hi-tech firm Rostec.
The announcement comes after the mayor of Kharkiv, a city in eastern Ukraine, was shot and critically wounded.
Hennadiy Kernes was recovering after an operation to repair damage to the chest and abdomen, but his life remained in danger, his office said.
Monday also saw pro-Russian separatists, whom Western nations accuse Moscow of supporting, seize a local government building in Kostyantynivka, a town to the south.
Separatists were also continuing to detain about 40 people in the town of Sloviansk, including journalists, pro-Kiev activists and 12 military observers from the Organisation for Security and Co-operation in Europe (OSCE), Ukraine's security service said.
At a special meeting of the OSCE's permanent council in Vienna, Russian ambassador Andrei Kelin said Moscow was taking "steps" to secure the observers' release. But the area around Sloviansk was very tense and it had been "extremely irresponsible" to send them there, he added.
The US and EU first imposed visa bans and asset freezes on a number of senior Russian officials and companies after Moscow annexed Crimea from Ukraine last month.
On Thursday, the White House said it was adding to its sanctions list "in response to Russia's continued illegal intervention in Ukraine and provocative acts that undermine Ukraine's democracy and threaten its peace, security, stability, sovereignty, and territorial integrity".
It accused Russia of "doing nothing to meet the commitments it made" at a meeting with Ukraine, the US and EU in Geneva on 17 April, which it said had included refraining from violence or provocative acts.
Russian Deputy Foreign Minister Sergei Ryabkov said Moscow's response would be "painful for Washington".
He told the Interfax news agency that the Kremlin was "disgusted" by the sanctions, which he said showed the White House had "completely lost touch with reality".
Among the seven government officials facing a US asset freeze and visa ban, two are seen as long-time allies:
Mr Sechin is a former KGB officer and deputy chief of staff during Mr Putin's first two presidential terms, when he masterminded Russia's energy policy
Mr Chemezov is described by the US as a trusted ally dating back to the 1980s
Alexei Pushkov is chairman of the committee of international affairs of Russia's lower house of parliament, the State Duma
Many of the 17 companies targeted are linked to individuals designated in the previous sanctions list. The latest measures also target some hi-tech exports that "could contribute to Russia's military capabilities".
Diplomats in Brussels also said European Union governments had reached a preliminary agreement to impose asset freezes and visa bans on another 15 people as part of expanded sanctions on Russia over its actions in Ukraine. The names are expected to be released on Tuesday, although more could still be added.
The developments came hours after Kharkiv's mayor was shot in the back while cycling on the outskirts of the city.
The head of the hospital where Mr Kernes was being treated, the Surgery Institute in Kharkiv, said he had suffered a "very serious wound", and that several organs had been damaged.
Valeriy Boyko said the threat to the mayor's life had not been eliminated, but that the bleeding had been stopped, his condition was stable and that doctors were treating him for shock.
Mr Kernes used to be a supporter of former pro-Moscow President Viktor Yanukovych. He then dropped his support for Mr Yanukovych in favour of a united Ukraine.