British Prime Minister Theresa May's spokesman dismissed the interview.
The BBC's Sarah Rainsford in Moscow described the interview as carefully choreographed and freaky, pointing out that in tone and content it matched the whole Russian response to the case - flat denial mixed with mockery.
London believes that Putin personally sanctioned the attack.
May rallied support from allies for coordinated expulsions of more than 150 Russian diplomats, prompting tit-for-tat retaliation from Moscow.
In a short clip, the two men claimed they were in the town for tourism, and that they may have been near Skripal's house by chance without realizing it, Reuters reported. Margarita Simonyan, RT's editor-in-chief, who conducted the interview, said on Twitter that "their answers are their responsibility" and it's up to viewers to decide whether to believe the men, who were "extremely nervous and sweating". When you go through the customs you have your luggage checked. "We came to you for protection, but this is turning into some kind of interrogation".
"How can the GRU allow their obedient officers to be ridiculed like this?" he added. She'd poured them some brandy "for courage".
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In the 25-minute video, the pair said they now fear for their lives, and hinted that British secret services want to bring them harm.
In the interview, they claimed to be in the fitness industry and said friends had suggested they visit "wonderful" Salisbury and to go sightseeing around Wiltshire.
United Kingdom authorities have pinned the poisoning on two Russian men they believe are intelligence officers, and charged them in their absence with conspiracy to murder, attempted murder and use of the nerve agent Novichok.
Scotland Yard published a series of photographs of the two men who, according to the investigation, were travelling around the country with passports issued in the names of Alexander Petrov and Ruslan Boshirov.
The two Russian men, who say they are both about 40, were identified last week as suspects by the British government.
"If the real perpetrations are found, I hope at least they (the British) will apologize to us", Petrov said. The two Russians entered the United Kingdom on an Aeroflot flight on March 2, departing March 4. They visited Salisbury twice by train on day trips - on the Saturday for reconnaissance and on the Sunday to poison Skripal. The victims recovered after treatment in the hospital.
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When asked to reveal personal details about themselves or explain why they were sharing a hotel room or taking trips together, Boshirov said: "Let's not pry into our private lives".
The men said they're partners in a business importing sports-nutrition supplements from Europe.
When the interviewer asked them whether they had Novichok or any poison with them, they emphatically said no.
Simonyan said the men had contacted her on her cellphone. "It's quite hard to come up with a good story as an alternative".
London pointed a finger at Russian President Vladimir Putin as "ultimately responsible" for the crime. United Kingdom authorities blamed two former Russian agents, Andrei Lugovoi and Dmitri Kovtun, for the murder.
Russian Federation has repeatedly denied any involvement.
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According to RT, the two men "sounded distressed" about being named by the United Kingdom as Russian intelligence agents alleged to have been involved in the poisoning. In 2017, the USA imposed sanctions on him for his role in the case.