Spermatozoa represent a tissue readily accessible for study after various exogenous perturbations. To characterize the lipid composition of monkey sperm and to establish a baseline from which dietary or pharmaceutical influences may then be evaluated, we collected semen samples from five rhesus monkeys by electroejaculation and analyzed the sperm for sterols, fatty acid composition, and the molecular species of the ethanolamine glycerophospholipids. Two sterols were identified: cholesterol, 41%, and desmosterol, 59% of total sterols. Desmosterol was found only in the free form. Cholesterol existed in three different forms: free, 60%; esterified, 20%; and sulfated, 20%. Docosahexaenoic acid (22:6, DHA) was almost the only n-3 fatty acid in sperm phospholipids, 24% of the total fatty acids. DHA was present mainly in phosphatidylcholine and phosphatidylethanolamine. Oleic and palmitic acids were the predominant monounsaturated and saturated fatty acids. The ethanolamine glycerophospholipids were separated into three subclasses: diacyl 49%, alkenylacyl 43%, and alkylacyl 8%. Thirteen molecular species were identified and quantified. The sn-1 position of these molecular species contained exclusively 16:0, 18:0, or 18:1. The sn-2 position contained n-3, n-6, and n-9, as well as saturated fatty acids. The molecular species having n-3 fatty acids in the sn-2 position contributed 43, 73, and 100% of the total in the diacyl, alkenylacyl, and alkylacyl subclasses, respectively. The presence of the unusual sterol, desmosterol, a cholesterol precursor not found in measurable quantities in any other tissue suggests an important functional and structural role for desmosterol in spermatozoa. The other unique lipids, cholesterol sulfate and the n-3 docosahexaenoic acid, may also have a significant role in the function of spermatozoa.