Washed cells of normal human ejaculates were incubated with [14C]arachidonic acid (20:4(n - 6] at 37 degrees C for 30-40 min and the main product was characterized as 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid by reverse phase, straight phase and chiral phase high performance liquid chromatography (HPLC) and by capillary gas chromatography-mass spectrometry. The biosynthesis of 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid from exogenous 20:4(n - 6) was inhibited by nordihydroguaiaretic acid and abolished by heat inactivation, but it appeared to be unaffected by the ionophore A23187 and Ca2+. Human spermatozoa were partly purified from contaminating material by the swim-up procedure and incubated with 14C-labelled 18:2(n - 6), 20:4(n - 6), 22:5(n - 6) and 22:6(n - 3) for 30-40 min at 37 degrees C. The main radiolabelled products, which were obtained in low yields, co-chromatographed with the Ls (n - 6)-hydroxy fatty acid of each substrate on reverse phase, straight phase and chiral phase HPLC. The (n - 6)-lipoxygenase was also present in ejaculates with oligozoospermia or azoospermia. The seminal fluid contains membrane-surrounded organelles (e.g., 'prostasomes' secreted by the prostate gland) and the (n - 6)-lipoxygenase was present and appeared to be relatively prominent in almost cell-free preparations of organelles of seminal fluid. The (n - 6)-lipoxygenase activity associated with the spermatozoa may thus be explained by the presence of prostasomes or other organelles, which may conceivably bind to the spermatozoon through hydrophobic interactions.