Double-strand breaks introduced into DNA in vivo have been shown to enhance homologous recombination in a variety of chromosomal and extrachromosomal loci in Saccharomyces cerevisiae. To introduce double-strand breaks in DNA at defined locations in mammalian cells, we have constructed a mammalian expression vector for a modified form of I-Sce I, a yeast mitochondrial intron-encoded endonuclease with an 18-bp recognition sequence. Expression of the modified I-Sce I endonuclease in COS1 cells results in cleavage of model recombination substrates and enhanced extrachromosomal recombination, as assayed by chloramphenicol acetyltransferase activity and Southern blot analysis. Constitutive expression of the endonuclease in mouse 3T3 cells is not lethal, possibly due to either the lack of I-Sce I sites in the genome or sufficient repair of them. Expression of an endonuclease with such a long recognition sequence will provide a powerful approach to studying a number of molecular processes in mammalian cells, including homologous recombination.