Seminal plasma inhibits human sperm from developing the ability to undergo the acrosome reaction. The inhibitory activity was identified as that of cholesterol on the basis of its solubility in organic solvents, its chromatographic behavior (adsorption, thin-layer, and gas chromatography), and its mass spectrum. Contrary to findings in other reports, no evidence for inhibitory proteins or peptides was found, and spermine was not an effective inhibitor. The inhibitory activity of untreated seminal plasma from individual ejaculates was highly correlated with the cholesterol content of the ejaculates (r = 0.96), suggesting that the amount of cholesterol determines the inhibitory activity of unfractionated seminal plasma. The inhibitory activity of unfractionated seminal plasma was significantly less, relative to the cholesterol content, than the activity of pure cholesterol, which is consistent with the idea that there are components in seminal plasma that partially counter the effect of cholesterol by promoting the development of acrosomal responsiveness.