To initially determine the effect that base-pair mismatch has on homologous recombination in mammalian cells, we have studied genetic recombination between thymidine kinase (tk) gene sequences from herpes simplex virus 1 and 2. These tk genes are approximately 81% homologous at the nucleotide level. We observed that, in mouse LTK- cells, intrachromosomal recombination between type 1 and type 2 tk sequences is reduced by a factor of at least 1000 relative to the rate of intrachromosomal recombination between homologous type 1 tk sequences. In sharp contrast, the rate of intermolecular or intramolecular extrachromosomal recombination between the heterologous tk sequences introduced by calcium phosphate or microinjection was reduced only by a factor of 3 to 15 compared with extrachromosomal homologous tk crosses. Our results suggest differences between the mechanisms of extrachromosomal and intrachromosomal recombination in mammalian cells.