Platelet activating factor (PAF) is a unique signaling phospholipid that has many biologic properties in addition to platelet activation. PAF roles in reproduction involve ovulation, fertilization, embryo development, implantation and parturition. It may also serve as a biomarker for normal sperm function. The presence of PAF receptor on the spermatozoa of 10 stallions was investigated by immunofluorescence microscopy. Statistical analysis revealed that the fluorescence intensity, FI (Mean+/-SEM), in the post- acrosomal region (FI= 2.60+/-0.15) was significantly higher (P< 0.01) than that in any other region of stallion spermatozoa. The effect of synthetic PAF on stallion spermatozoal motility, capacitation, and the acrosome reaction (AR) were evaluated. Treatment of 10 stallion semen samples with 10 â 4 to 10 â 13 M PAF resulted in statistically significant differences in motility and capacitation (r2 = 0.81 and 0.83 respectively). The concentration of PAF, incubation time and their interaction were highly significant (P< 0.01) for their effect on motility. Concentrations of PAF ranging from 10-9 to10-11 M were able to induce capacitation. Following capacitation in vitro with PAF, and induction of the acrosomal reaction by progesterone, transmission electron microscopy (TEM) was conducted on the spermatozoa of 3 stallions, to detect the true AR. Differences in PAF concentrations were highly significant as indicated by R-square (for intact: 97.2, reacted: 89.8, and for vesiculated: 98.1). Treating spermatozoa from 3 stallions with the PAF antagonist FR-49175 inhibited calcium release and fluorescence intensity with a median inhibitory concentration (IC50) of 10-7.5 M (r2=0.82, P<0.01) and 10-8 M (r2=0.92, P<0.01) respectively, suggesting a receptor mediated process for the mechanism of action of PAF. Although the exact mechanisms of PAF action on equine spermatozoa remain unclear, it is widely reported that PAF acts by a receptor-mediated mechanism and that the PAF receptor is a member of the family of G-protein coupled receptors with phospholipase C as the effector. Since the limited success in equine ART (e.g. IVF) is in part due to lack of efficient treatment of stallion spermatozoa for capacitation, PAF may be useful to help capacitate stallion spermatozoa. Without proper capacitation, spermatozoa are unable to initiate the acrosome reaction which is a prerequisite for fertilization.