The highly conserved RAD51 protein has a central role in homologous recombination. Five novel RAD51-like genes have been identified in mammalian cells, but little is known about their functions. A DNA damage-sensitive hamster cell line, irs3, was found to have a mutation in the RAD51L2 gene and an undetectable level of RAD51L2 protein. Resistance of irs3 to DNA-damaging agents was significantly increased by expression of the human RAD51L2 gene, but not by other RAD51-like genes or RAD51 itself. Consistent with a role for RAD51L2 in homologous recombination, irs3 cells show a reduction in sister chromatid exchange, an increase in isochromatid breaks, and a decrease in damage-dependent RAD51 focus formation compared with wild type cells. As recently demonstrated for human cells, we show that RAD51L2 forms part of two separate complexes of hamster RAD51-like proteins. Strikingly, neither complex of RAD51-like proteins is formed in irs3 cells. Our results demonstrate that RAD51L2 has a key role in mammalian RAD51-dependent processes, contingent on the formation of protein complexes involved in homologous recombination repair.