HIV-1 virions contain two reverse transcriptase polypeptides that have apparent molecular weights of 66 and 51 kDa. The 51-kDa form lacks the carboxy-terminal sequences found in the 66-kDa form, and is believed to be a proteolytic digestion product. We have treated purified 66-kDa reverse transcriptase with viral and nonviral proteases. The digestion products were characterized by their ability to react with monoclonal antibodies known to recognize particular segments of the HIV-1 reverse transcriptase. The approximate location of the segments recognized by the monoclonal antibodies was determined by testing the ability of the antibodies to recognize a series of amino- and carboxy-terminal-deleted forms of HIV-1 reverse transcriptase. The segments recognized are not uniformly distributed along the primary amino acid sequence of HIV-1 reverse transcriptase. We suggest that these segments are probably on the surface of the properly folded form of reverse transcriptase. Of the tested proteases, only the viral protease was able to cleave the 66-kDa form to the 51-kDa form without producing additional cleavage products, suggesting that the viral protease cleaves the 66-kDa protein to the 51-kDa form in virions.