The reverse transcriptase of HIV-1 (AIDS virus) is characterized by the presence of two highly immunogenic proteins of 66 and 51 kD known to be enzymatically active as a complex p66/51. Using an activity gel procedure that allows identification of catalytic polypeptides in situ after PAGE in denaturing conditions, we visualized two major active bands of 66 and 51 kD of reverse transcriptase from highly purified preparations of HIV-1. We show that both p66 and p51 are enzymatically active. An additional active band was also associated with a 165 kD polypeptide, representing about 2-4% of total activity and possibly corresponding to the putative gag-pol precursor. In H9-infected cells the 66 kD active band became visible 70 hours after infection. These studies show that the two major forms of reverse transcriptase (66 and 51 kD) of HIV-1 are independently active and that a higher Mr form of 165 kD is also enzymatically active.