The normal reverse transcription of retroviral RNA is a complex process which depends on the orchestration of several steps throughout the virus life cycle. During the assembly of retroviruses, reverse transcriptase (RT) is directed into the virion as a component of the Gag-Pol polyprotein. In the maturation of the Gag-Pol polyprotein of human immunodeficiency virus type 1 (HIV-1), cleavage by the viral protease occurs during viral budding. After infection, reverse transcription of viral RNA into double-stranded DNA is completed in the cytoplasm of the infected cell. In this study, the processing and reverse transcription of HIV-1 have been examined by separate expression of mature HIV-1 RT and proviral molecules bearing RT mutations. The effects of RT expression in trans during virion release and after viral entry were investigated. Constitutive expression of HIV-1 RT was established in CD4- and non-CD4-expressing cells via the coexpression of its individual subunits, and three HIV-1 RT mutant constructs were generated. The results indicate that a bona fide RT trans complementation does not occur during virion release or after infection. However, after infection of an RT-expressing cell with a high titer RT-defective virus, intracellular reverse transcription can be detected.