The RecA protein is the central enzyme in prokaryotic recombination. It catalyzes pairing and strand exchange between homologous DNA molecules, and functions in both DNA repair and genetic recombination. The RecA-like proteins Rad51 and Dmc1 of yeast are both required for meiotic recombination and the former is also necessary for repair of double-strand breaks in vegetative cells. Genes encoding Rad51 homologs have been isolated recently from several higher eukaryotes. This paper describes the isolation and molecular characterization of a genomic DNA fragment from Drosophila melanogaster containing the coding sequence for a RecA-like protein. This protein exhibits strong sequence homology with the Rad51 proteins of budding yeast, fission yeast, chickens, mouse and humans, and slightly less (but still strong) homology with yeast Dmc1. Both in situ hybridization and Southern analysis indicate that the Rad51 gene is present only once per genome in Drosophila (at 99D on chromosome arm 3R). However, there are at least three other fragments that cross-hybridize strongly at low stringency. RNA blotting analysis detects a single transcript of about 1.35 kb that is present throughout development at low levels. Transcript levels are induced at least tenfold in ovaries, as measured by RNase protection analysis, suggestive of a role in female meiosis. Transcript levels are significantly lower in testes than in bulk RNA of adult males, however, indicating that Rad51 may be repressed in meiosis of Drosophila males.