The State Prosecutor's Office has not divulged details of the inquiry into allegations of negligence by public officials, including prosecutors, one of eight probes into the crash.
The ruling conservative Law and Justice (PiS) party has said that Polish officials were not in Russia in the crucial hours after the crash and did not ask to be at the autopsies.
Tusk, currently European Council president, was Poland's prime minister at the time of the crash. The health minister, the justice minister, the foreign minister and prosecutors of the day have already been heard in the inquiry.
A crowd of supporters and opponents met Tusk before his hearing in Warsaw, much as before his April hearing as a witness in a spying probe.
Ahead of the hearing, the leader of Poland's ruling Law and Justice (PiS) party, Jarosław Kaczyński, said Tusk should be concerned.
Tusk said: "That affirms the concerns of many who think Jarosław Kaczyński wants the justice system to answer to him".
In April 2016, the State Prosecutor's Office took over investigations into the 2010 Smolensk disaster, which killed 96 people, including then-President Lech Kaczyński – Jarosław Kaczyński's twin – and many top military and political figures.
The prosecution started to exhume all the bodies that were neither cremated nor previously exhumed following allegations of errors in Russian autopsy reports.
The prosecution last week said that nearly half of the DNA tests on exhumed bodies so far have shown that body parts, and even entire sets of remains, were mixed up.
Parts of as many as eight different people were found in a single coffin.
Meanwhile, a government commission set up after PiS came to power in 2015 has claimed that the presidential plane was probably brought down by an explosion, and that Russian air traffic controllers deliberately misled Polish pilots about their location, as they approached the runway of the Smolensk military airport in western Russia on 10 April 2010.
PiS has long challenged an official report into the crash issued by the previous Polish government which cited a catalogue of errors on the Polish side, while also pointing to errors made by Russian staff at the control tower of Smolensk Military Airport.
A Russian report placed all the blame on the Poles. (vb/pk)