The Saccharomyces cerevisiae strand exchange protein 1 (Sep1; also referred to as Xrn1, Kem1, Rar5, or Stp beta) catalyzes the formation of hybrid DNA from model substrates in vitro. The protein is also a 5'-to-3' exonuclease active on DNA and RNA. Multiple roles for the in vivo function of Sep1, ranging from DNA recombination and cytoskeleton to RNA turnover, have been proposed. We show that Sep1 is an abundant protein in vegetative S. cerevisiae cells, present at about 80,000 molecules per diploid cell. Protein levels were not changed during the cell cycle or in response to DNA-damaging agents but increased twofold during meiosis. Cell fractionation and indirect immunofluorescence studies indicated that > 90% of Sep1 was cytoplasmic in vegetative cells, and indirect immunofluorescence indicated a cytoplasmic localization in meiotic cells as well. The localization supports the proposal that Sep1 has a role in cytoplasmic RNA metabolism. Anti-Sep1 monoclonal antibodies detected cross-reacting antigens in the fission yeast Schizosccharomyces pombe, in Drosophila melanogaster embryos, in Xenopus laevis, and in a mouse pre-B-cell line.