Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=1-O-alkylacetyl-sn-glycero-3-phosphocholine) is a signalling phospholipid that has many biological properties in addition to platelet activation. PAF receptors have been detected on stallion spermatozoa; therefore, the aim of this study was to evaluate the effect of synthetic PAF on the motility, capacitation and the acrosome reaction of stallion spermatozoa. Treatment of ten stallion semen samples with 10-4-10-13 mol PAF I-1 resulted in significant differences in motility and capacitation (r2 = 0.81 and 0.83, respectively). Statistical analysis indicated that PAF also has an effect on acrosome reaction (r2 = 0.20). PAF concentrations, incubation time and their interaction had a highly significant (P < 0.01) effect on motility. After capacitation in vitro with PAF, and induction of the acrosome reaction by progesterone, transmission electron microscopy was conducted on the spermatozoa of three stallions to detect the true acrosome reaction. Differences in PAF concentrations were highly significant (r2 for intact: 97.2; reacted: 89.8; and vesiculated: 98.1). The results indicate that a lower concentration of PAF enhances motility and induces capacitation of stallion spermatozoa, whereas a higher concentration of PAF induces the acrosome reaction.