The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.