Palestinian medics said the strike, which killed Khalid Abu Salima, 33, happened on Sunday evening.
Abu Salima was a member of Hamas' military wing - the Izz al-Din al-Qassam Brigades.
His car took a direct hit from a rocket fired by an Israeli aircraft on al-Jalah street in the heart of the city, witnesses said, adding that the aircraft may have been an unmanned drone.
A spokesman for the Israeli military had no immediate comment.
Hamas vowed to avenge the death of Abu Salima, whom it described as a senior field commander of the group's armed wing which has been behind scores of attacks against Israel over the past decade.
"It was a clear assassination of one of our mujahidiin (fighters) ... Hamas will be able and capable of teaching the enemy a painful lesson," said Mushir al-Masri, a Hamas spokesman.
Three members of the al-Aqsa Martyrs' Brigades, an offshoot of Palestinian leader Yasir Arafat's Fatah faction, were killed in a similar Israeli air strike on their vehicle in the northern West Bank last Monday.
Israel's policy of targeting resistance fighters and politicians has been condemned by human rights groups and many members of the international community.
Earlier in the day Abd Al-Karim Abu Lihya, 20, was killed and three other fellow fighters were wounded in Gaza as they reportedly tried to plant explosives near the Karni border crossing into Israel.
An Israeli military source said several Palestinians were spotted approaching a security fence near Karni.
West Bank siege
Meanwhile, in the West Bank, according to Aljazeera's correspondent in Jenin, Ali al-Sumudi, a raid began in the early hours with Israeli military vehicles entering al-Sikka neighbourhood via the southern gate of the town.
The forces who conducted house-to-house searches said they were looking for resistance fighters, the correspondent said.
The West Bank town of Jenin has
been a target of frequent raids
An exchange of gunfire between Israeli troops and fighters from al-Aqsa Martyrs Brigades and al-Quds Brigades lasted for about two hours.
The town has been turned into a prison due to the closure imposed four days ago, the correspondent said.
Residents, including the sick, have been prevented from entering or leaving the town.
Israeli occupation forces have also fired at ambulances, warning them against moving around in the city or transferring patients to hospitals.