Studies were conducted on platelet-activating factor (PAF) in male reproductive organs. Total lipids were extracted from the seminal vesicles, and PAF was purified by silicic column chromatography, thin-layer chromatography, and HPLC. PAF activities in the seminal vesicles of guinea pigs and rats as measured by assay of platelet aggregation were 0.26 and 0.25 ng PAF/mumol original phospholipids. These activities were inhibited by the specific PAF antagonists CV6209 and L652,731. In guinea pigs, PAF activity decreased to 50% and 31% of the normal level 2 wk and 4 wk, respectively, after castration. The activity increased to 136% of the normal level on administration of 1 mg of testosterone propionate every day for 1 wk beginning 2 wk after castration, but decreased to 74% of the control level on administration of vehicle. Furthermore, administration of 100 IU of hCG every other day to guinea pigs resulted in increased activity, to 132% (p < 0.05) and 154% (p < 0.01) of the activity in vehicle-treated animals on Days 1 and 3, respectively. Electron microscopic studies showed changes in the rough endoplasmic reticulum of the epithelial cells of the seminal vesicles in castrated guinea pigs. (The rough endoplasmic reticulum is involved in PAF biosynthesis.) PAF activity was also demonstrated in tissues of the testis, epididymis, and vas deferens of guinea pigs and rats, and in the prostate of rats. Thus PAF may play important physiological roles in male reproductive organs.