Both the full-length and spliced RNA species of HIV-1 possess the necessary cis-acting elements including the primer binding site (PBS), the polypurine tract (PPT), as well as the 5' R and 3' R regions that are needed for their conversion to double-stranded cDNA through reverse transcription. Since measurable amounts of spliced viral RNA molecules can be detected within virus particles, we have examined the potential for reverse transcription of such virion-associated spliced viral RNA upon infection of permissive cells. Analysis of viral cDNA species by PCR and DNA sequencing not only led to the identification of viral DNA molecules that were reverse transcribed from full-length viral RNA, but also DNA molecules that displayed the same nucleotide sequences as those found in spliced viral RNA, except that the former harbored the complete long terminal repeats (LTR), a feature that distinguishes proviral DNA from viral genomic RNA. Further studies revealed various types of cDNA species that resemble the spliced viral RNA encoding each of the env, tat, rev, or nef genes, of which the nef cDNA represents the majority. Therefore, spliced HIV-1 RNA molecules must have been reverse transcribed along with full-length viral RNA during infection.