We used previously characterized spleen necrosis virus-based retroviral vectors and helper cells to study the strand transfers that occur during the reverse-transcription phase of a single cycle of retroviral replication. The conditions used selected only for formation of an active provirus rather than for expression of multiple drug resistance markers. In nonrecombinant proviruses the minus- and plus-strand DNA primer transfers were almost completely intramolecular. However, as previously reported, recombinant proviruses contained approximately equal proportions of inter- and intramolecular minus-strand DNA primer transfers. Thus, we conclude that in the absence of recombination, one molecule of retroviral RNA is sufficient for viral DNA synthesis. Large deletions and deletions with insertions were detected primarily at a limited number of positions which appear to be hot spots for such events, the primer binding site and regions containing multiple inverted repeats. At these hot spots, the rate of deletions and deletions with insertions visible with PCR was about 10% per genome per replication cycle. Other deletions and deletions with insertions (detectable with PCR) occurred at a rate of about 0.5%/kb per replication cycle. Crossovers occurred at a rate of about 6%/kb per replication cycle under single-selection conditions. This rate is comparable to the rate that we reported previously under double-selection conditions, indicating that retroviral homologous recombination is not highly error prone. The combined rates of deletions and deletions with insertions at hot spots (10% per genome per replication cycle) and other sites (0.5%/kb per replication cycle) and the rate of crossovers (6%/kb per replication cycle) indicate that on average, full-size (10-kb) type C retroviruses undergo an additional or aberrant strand transfer about once per cycle of infection.