Phospholipase D1 (PLD1) is an important enzyme involved in lipid-mediated signal transduction and membrane dynamics in eukaryotes. PLD1 preferentially hydrolyzes phosphatidylcholine to phosphatidic acid. This potent second messenger is involved in cytoskeletal reorganization, secretion, and membrane trafficking in eukaryotic cells. In Saccharomyces cerevisiae, PLD1 is involved in polarized growth and morphogenesis during pheromone response and sporulation. The presence of a PLD activity in Schizosaccharomyces pombe is demonstrated. PLD activity was able to hydrolyze a fluorescently labeled analog of phosphatidylcholine and was capable of performing the transphosphatidylation reaction characteristic of PLDs. Schizosaccharomyces pombe PLD activity was unaffected by phosphatidylinositol 4,5 bisphosphate (PIP(2)), but was slightly stimulated by oleate. PLD activity was shown to increase when the S. pombe cells underwent mating and sporulation. Here, we also report the molecular cloning of the first phospholipase D isoform from an S. pombe genomic DNA library (EMBL accession no. FN547388). Comparisons of three divergent yeasts, S. pombe, S. cerevisiae, and Candida albicans, with respect to the PLD enzymes revealed differences in regulation by oleate and PIP(2). Even with high homology in the protein sequences between the PLD1 enzymes of S. cerevisiae, C. albicans, and S. pombe, there was variation with the effects of the regulators.