The closely related B. subtilis bacteriophages SPO1 and SP82 have similar introns inserted into a conserved domain of their DNA polymerase genes. These introns encode endonucleases with unique properties. Other intron-encoded ''homing'' endonucleases cleave both strands of intronless DNA; subsequent repair results in unidirectional gene conversion to the intron-containing allele. In contrast, the enzymes described here cleave one strand on both intron-containing and intronless targets at different distances from their common intron insertion site. Most surprisingly, each enzyme prefers DNA of the heterologous phage. The SP82-encoded endonuclease is responsible for exclusion of the SPO1 intron and flanking genetic markers from the progeny of mixed infections, a novel selective advantage imparted by an intron to the genome in which it resides.