Retroviruses convert their RNA genome into a DNA form by means of reverse transcription. According to the current model of reverse transcription, two strand transfer reactions are needed to synthesize a full-length DNA genome. Because reverse transcription is initiated close to the 5' end of the RNA genome, the first strand transfer translocates the minus-strand cDNA to the 3' end of the viral genome. This jump is facilitated by the presence of a perfect repeat element (R) at both ends of a retroviral genome. Strand transfer has been extensively studied in in vitro systems with purified reverse transcriptase enzyme (RT) and nucleic acid donor and acceptor templates. In this study, we set out to test several parameters of the strand transfer reaction as it occurs in cells infected with the human immunodeficiency virus (HIV-1). We constructed mutant HIV-1 genomes with 3' R acceptor sequences that were specifically altered either in length or structure. Analysis of the replication characteristics of the mutant viruses indicates that repeats much shorter than the wild-type 97-nucleotides R region can efficiently act as acceptors during reverse transcription. Furthermore, the introduction of excessively stable hairpin structures within the 3' R element did only marginally affect the strand transfer efficiency. We also analysed the DNA forms inherited upon infection of cells with HIV-1 templates with multiple 3' R copies. These experiments indicate that various 3' R repeats can serve as acceptor during minus-strand DNA transfer.