The bacteriophage T4 segE gene encoding site-specific endonuclease lies between the hoc.1 and uvsW genes. The similar region of T-even-related phage RB30 lacks the segE gene. Here we demonstrate that the phage T4 segE gene is inherited preferably by progeny of mixed infection with RB30. The preferred inheritance of the segE gene depends on its own expression and is based on a non-reciprocal homologous recombination event providing the transfer of the gene from the segE-containing to the segE-lacking allele. The SegE endonuclease cleaves DNA in a site located at the 5' end of the uvsW gene in the RB30 genome. The T4 DNA is also cleaved by the enzyme, but less efficiently. The cleavage at the RB30 site appears to initiate the observed conversion, which is stimulated by DNA homology and accompanied by co-conversion of flanking markers. Our findings provide a novel example of endonuclease-dependent generation of genetic variation in prokaryotes.