Homologous recombination (HR) is a precise mechanism for repairing harmful DNA double-strand breaks. The process has been extensively studied in microbial species leading to identification of the major proteins, HR models and homology requirements. Much less is known about HR in mammalian systems, especially early HR events. Our laboratory has recently devel-oped an assay that detects the new DNA synthesis that accompanies the early homology search and strand invasion steps of HR (the 3’ extension assay). The hypothesis that homology require-ments for the early steps of HR may differ from those identified in other HR assays was tested. Plasmids bearing varying amounts of homology to the chromosomal immunoglobulin μ target locus gene were constructed and tested in the 3’ extension assay. The homology require-ments for the 3’ extension assay were somewhat lower than might be expected based on other HR assays. An approximately linear relationship between homology length and 3’ extension was also established on each side of the double-strand break. The effect of excess Rad51, an essential protein involved in early HR, was also measured with respect to homology, leading to the dis-covery that increased Rad51 resulted in an increase in 3’ extension events independent of ho-mology. In summary, 3’ extension generates a potentially unstable, short-lived HR intermediate that has less dependence on homology than a completed HR product. Homology plays a role in the initiation of HR, but it may be more important in the stabilization of the intermediate than the actual generation of the early HR product detected in the 3’ extension assay. / CIHR