Many single-celled eukaryotes produce prostaglandin-like molecules, but these have not been absolutely verified by mass spectrometry. We have isolated, and identified by liquid chromatography-tandem mass spectrometry, authentic prostaglandin E(2) from Cryptococcus neoformans. Cyclooxygenase inhibitors did not inhibit prostaglandin synthesis, and the cryptococcal genome lacks a cyclooxygenase homolog. Thus, novel enzymes must exist.