Human spermatozoa are associated with arachidonate 15-lipoxygenase activity. This activity could be due to 15-lipoxygenase in small organelles (prostasomes), which are known to bind hydrophobically to germ cells. This possibility was assessed by separating prostasomes from human spermatozoa by differential centrifugation and purifying them by gel filtration (Sephadex G-200). Purified prostasomes metabolized [1-14C]arachidonic acid to 15(S)-hydroxyeicosatetraenoic acid as determined by reverse phase and by chiral phase HPLC and by gas chromatography–mass spectrometry. Biosynthesis of prostaglandins could not be detected, but the prostasomes contained trace amounts of the four major E prostaglandins of human seminal fluid (3.6 nmol mg−1 of prostasomal protein). Arachidonic acid 15-lipoxygenase has recently been implicated in the acrosome reaction of bull spermatozoa and it may have a similar function in the acrosome reaction of human spermatozoa.