Repair of all 12 single-base mismatches in recombination intermediates was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks in regions of shared homology. Recombination was predicted to occur via single-strand annealing, yielding heteroduplex DNA (hDNA) with a single mismatch. Nicks were expected on opposite strands flanking hDNA, equidistant from the mismatch. Unlike studies of covalently closed artificial hDNA substrates, all mismatches were efficiently repaired, consistent with a nick-driven repair process. The average repair efficiency for all mispairs was 92%, with no significant differences among mispairs. There was significant strand-independent repair of G-T --> G-C, with a slightly greater bias in a CpG context. Repair of C-A was also biased (toward C-G), but no A-C --> G-C bias was found, a possible sequence context effect. No other mismatches showed evidence of biased repair, but among hetero-mismatches, the trend was toward retention of C or G vs. A or T. Repair of both T-T and G-T mismatches was much less efficient in mismatch repair-deficient cells (approximately 25%), and the residual G-T repair was completely biased toward G-C. Our data indicate that single-base mismatches in recombination intermediates are substrates for at least two competing repair systems.