Repair of heteroduplex DNA, generated between two interacting DNA molecules during homologous recombination, has been implicated as a contributing factor in the process of gene conversion. To assess patterns of heteroduplex repair in mammalian cells, we constructed 13 different heteroduplexes from simian virus 40 wild-type and deletion mutant DNAs. Each heteroduplex contained one or multiple single-stranded loops in the intron of the gene for large tumor antigen, which is not essential during lytic infection. After transfection into cultured monkey cells, cellular repair was evaluated by restriction analysis of the amplified viral progeny from 1123 individual plaques, each representing the clonal expansion of a single repair event. Single-stranded loops were corrected prior to replication with an overall efficiency of 90%. At the position of a loop, one of the two heteroduplex strands served as a template for accurate repair 98% of the time. Repair of single-stranded loops was biased nearly 2 to 1 in favor of the strand without the loop. The efficiency, accuracy, and strand bias of repair were unaffected by loop size within the tested range, which was 25-247 nucleotides. The excision tract associated with repair of single-stranded loops rarely exceeds 200-400 nucleotides in length.