The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity. Previous studies have shown that vif mutant HIV-1 virions are defective in their ability to synthesize proviral DNA in vivo. Here, we examine the role of Vif in viral DNA synthesis in the endogenous reverse transcriptase (RT) reaction, an in vitro assay in which virions synthesize viral DNA by using endogenous viral RNA as a template. vif mutant virions showed a significant reduction in endogenous RT activity despite similar levels of exogenous RT activity. Analysis of the viral DNA products on agarose gels demonstrated that this reflects reduced synthesis of short minus- and plus-strand DNA products in addition to those of full genomic length. Quantitative PCR analysis of endogenous reverse transcription provided further evidence for reduced formation of both initial and completed reverse transcripts. Vif had no effect on genomic RNA dimerization or the stability of the RNA dimer linkage. These results suggest that Vif is important for an early event after virus entry but preceding or during the early stages of viral DNA synthesis. This may be due to an intrinsic effect on reverse transcription or a preceding postentry event(s), such as virion uncoating or disassembly of the virion core. Drugs targeted to Vif function may provide a new therapeutic approach to inhibiting HIV-1 reverse transcription.