The 202 people who were returned from Greece to Turkey on Monday did not ask for asylum, in spite of having been informed of their right to do so.
This has emerged after Turkey said it is ready to take another 200 migrants deported from the Greek islands on Wednesday.
Some rights groups said there was confusion as to whether those migrants who apply for asylum in Greece can stay in Greece or will be processed and then sent back to Turkey, even if they have an asylum case.
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But, on Tuesday, the European Commission sought to clarify the situation by issuing a statement through its deputy chief spokesperson.
She said, "First, we are not speaking about 'deportations' but of 'returns' on the basis of an existing legal framework."
She added, "Those who apply for asylum in Greece, have the right to have their applications examined in Greece and can stay in Greece until the application has been examined.
"Only if Greece decided to make use of the 'safe third country' or 'first country of asylum' principle (provided for in EU and their national law), can it send back asylum seekers without examining their application."
In this case, the Commission says that their application would be examined in Turkey "as they had already been previously granted temporary protection there or because they have access to the asylum procedure there."
The first group of 202 migrants, mostly Pakistani and Afghan, were shipped back to Turkey on Monday under an agreement which will see Ankara take back all migrants and refugees who cross the Aegean to enter Greece illegally.
In return, the EU will take in thousands of Syrian refugees directly from Turkey and reward it with money, visa-free travel and progress in its EU membership negotiations.
The Commission points out that those who were returned on Monday did not ask for asylum in spite of having been informed of their right to do so.
"Yesterday," added the spokesperson, "we saw the return of irregular migrants who had chosen not to apply for asylum.
"More generally, the Commission has made clear that no one in need of international protection will be returned to Turkey who does not already benefit or will receive protection in Turkey."
Rights groups and some European politicians have challenged the legality of the EU-Turkey deal, questioning whether Turkey has sufficient safeguards in place to defend refugees' rights and whether it can be considered safe for them.
The United Nations refugee agency UNHCR has stopped transporting arrivals to and from the Moria camp on Lesbos, initially set up to register arrivals but which has since become what it calls a "detention center".
Meanwhile, the EU announced on Tuesday that it is to allocate €100m development package for Sudan to address root causes of irregular migration and forced displacement.
It will be implemented under the EU Emergency Trust Fund for Africa, set up last year to tackle instability and the root causes of irregular migration and forced displacement.