The 45 migrants, who a police source said were all Pakistani nationals, departed from the island of Lesbos on the Turkish ferry Lesvos.
Three activists were arrested after clinging onto the ferry's anchor to prevent it from leaving.
Another 30 people gathered at the port of Lesbos, chanting "Stop deportations", "EU shame on you" and "Freedom for the refugees".
Another group of around 80 migrants is expected to leave Lesbos later in the day.
According to the Greek government, the operation concerns people who have not requested asylum in Greece.
A first batch of 202 migrants were sent to Turkey from the islands of Lesbos and Chios on Monday, sparking a flurry of asylum demands and forcing Greek authorities to delay further expulsions as they have to examine the demands individually.
Greek officials now expect a two-week delay while authorities examine the first of those asylum applications.
Fear of deportation has prompted acts of desperation in some of the camps where migrants are held.
Yesterday, some 150 migrants broke out of a camp on the island of Samos but were later persuaded to return, the government source said.
Dozens of migrants on Samos and Lesbos also claim to have begun a hunger strike to prevent their expulsion and demand the reopening of borders shut by Balkan nations last month.
One Pakistani hunger striker collapsed in Moria, the main camp on Lesbos, and was taken for treatment by medical charity Medecins du Monde.
Another Pakistani on Samos, named Ali, told AFP that a dozen of his countrymen were on hunger strike there.
"They told us to apply for asylum in Greece and that if not, we are going to be sent back to Turkey.
Under the terms of the EU-Turkey deal, all "irregular migrants" arriving on the Greek islands from Turkey since March 20 face being sent back, although the accord calls for each case to be examined individually.
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