Trichomonas vaginalis and Tritrichomonas foetus grown in a fetal calf serum-based culture medium, contained as major lipids (i.e., >10% of total) cholesterol, phosphatidylethanolamine and phosphatidylcholine. T. vaginalis also contained sphingomyelin and T. foetus glycophosphosphingolipids. The culture medium contained (>10%) cholesterol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine. The fatty acyl groups of these major lipids of the trichomonads and the culture medium were similar. Those present in amounts >5% of the total fatty acyl groups for a given lipid were myristic, palmitic, hexadecaenoic, stearic, oleic, linoleic, arachidonic and docosahexaenoic. When the trichomonads were exposed to radiolabeled lipids and lipid precursors, [14C]-labeled acetate and potential acetate precursors (glucose, threonine) were poorly incorporated and failed to label the fatty acyl groups of the trichomonad lipids. [14C]-labeled, C12-C22 saturated and unsaturated fatty acids were incorporated, unaltered, into phosphoglycerides and sphingolipids (sphingomyelin and glycophosphosphingolipids), but not into cholesteryl esters or triacylglycerols. Phosphoglycerides were preferentially labeled with unsaturated fatty acids and sphingolipids with saturated ones. This information inferred that the trichomonads: 1) were unable to biosynthesize fatty acids de novo, 2) took up unesterified fatty acids from the culture medium and used them in phosphoglyceride and sphingolipid biosynthesis and/or turnover, 4) did not use unesterified fatty acids in the biosynthesis or turnover of cholesteryl esters or triacylglycerols. Phosphatidylcholine and phosphatidylethanolamine, with [14C]labeled fatty acyl groups, and sphingomyelin, with 14C-labeled choline, were incorporated by the trichomonads. The phospholipids strongly labeled phosphoglycerides and sphingolipids, but not triacylglycerols, while the radioactivity of sphingomyelin [14C]choline remained associated solely with trichomonad sphingomyelin. Triacylglycerol, with 14C-labeled fatty acyl groups, was also incorporated, and labeled phosphoglycerides and sphingolipids. The results of those experiments suggested that trichomonads: (1) could take up culture medium phospholipids and triacylglycerols; (2) actively deacylated and reacylated phospholipids, but not triacylglycerols; (3) hydrolyzed exogenous triacylglycerols and used their fatty acyl groups for phospholipid acylations. Radiolabeled acetate, mevalonate and squalene were not incorporated into trichomonad cholesterol or cholesteryl esters. [14C]Cholesterol was incorporated unaltered, but was not esterified. Cholesteryl oleate, 14C-labeled in the fatty acyl group, was incorporated, and labeled the phosphoglycerides but not the sphingolipids, while cholesteryl oleate, 3H-labeled in the cholesterol moiety labeled only cholesterol. The results of these experiments implied that trichomonads: (1) were unable to biosynthesize cholesterol de novo; (2) were able to incorporate cholesterol from the culture medium, but unable to alter its structure or to esterify it; (3) were able to incorporate cholesteryl esters, to hydrolyze them and to use their fatty acyl groups for phospholipid acylations. © 1990.