The bacteriophage UvsX protein is a "strand transferase" that promotes the pairing of homologous single and double-stranded DNAs. The efficiency of UvsX protein-mediated joint molecule formation between supercoiled duplex DNA and oligonucleotides is shown to have a sharp dependence on the degree of homology. The reaction proceeded efficiently with oligonucleotides containing 32 homologous positions but not with oligonucleotides containing only 24 homologous bases. This was shown to reflect an intrinsic homology requirement for the formation of stable joint molecules and was not caused by poor binding of the protein to short single-stranded DNAs. Even a single mismatch located in the middle of a region of 40 homologous nucleotides had a detectable effect on the efficiency of pairing. An in vitro recombinationally initiated DNA synthesis reaction that mimics the "secondary mode" of phage T4 DNA replication exhibited the same homology dependence.