Platelet-activating factor (PAF), a potent lipid mediator of inflammation, has been shown to play a role in both the implantation and viability of mammalian embryos. We examined whether human and mouse spermatozoa release PAF during in vitro incubation and assessed the effect of exogenous PAF and the PAF receptor antagonist WEB 2086, a thieno-triazolodiazepine, on mouse in vitro fertilization (IVF) rate. PAF biological activity was detected in 11 samples of leukocyte-free, purified human spermatozoa (28 pg PAF/106 cells/24 hr) and 5 samples of epididymal mouse spermatozoa (7.8 pg PAF/106 cells/3 hr). Exogenous PAF (10-8 and 10-6 M) increased (p <0.01) the fertilization rate 2- and 3-fold, respectively of mouse oocytes by mouse epididymal spermatozoa. 10-4 M PAF, however, reduced sperm motility and decreased (p < 0.05) the fertilization rate. 10-6 M WEB 2086, decreased IVF to approximately 50% of the control fertilization rate (42% vs. 89%). WEB 2086 treatment also promoted the attachment of supernumerary spermatozoa to both fertilized and unfertilized oocytes. The fertilization rate in the presence of WEB 2086 returned to control levels when zona-pellucida-free oocytes were employed, indicating that WEB 2086 did not interfere with the spermatozoal acrosome reaction. These data suggest that PAF, of spermatozoal origin, may be important in mammalian fertilization. © 1990.