In trypanosomatids, little is known about the biosynthetic pathways involved in the metabolism of ethanolamine. In an attempt to clarify this point, an exhaustive analysis of the chloroform:methanol extract of T. cruzi trypomastigotes metabolically labeled with [14C]ethanolamine, in comparison with the lipids from [3H]palmitic acid-incorporated parasites, was performed. In both cases, phosphatidylethanolamine and lysophosphatidylethanolamine were detected, while phosphatidylcholine and lysophosphatidylcholine were only labeled with the fatty acid precursor. However, dimethylphosphatidylethanolamine was isolated from parasites labeled with the base precursor, indicating the ability of trypanosomes to methylate phosphatidylethanolamine to dimethylphosphatidylethanolamine. Fatty acids of the labeled phospholipids were analyzed by reverse-phase thin-layer chromatography and fluorography. Interestingly, phospholipids from the trypomastigote stage show palmitic acid (C16:0) and stearic acid (C18:0) as the only labeled components. The same saturated fatty acids were found free and as components of the radioactive triglycerides. No unsaturated fatty acids were detected, in accordance with the results obtained with inositolphospholipids. Conversely, when the fatty acids of phospholipids purified from nonlabeled parasites were analyzed by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry, C18:1 was also detected. A striking finding was the presence of a considerable amount of free lignoceric acid (C24:0). Also, the C24:0 fatty acid was identified in the triglyceride fraction and as a component of phosphatidylcholine. The limited capacity of trypomastigote forms to elongate fatty acids was determined. In contrast with the results reported for other noninfective forms of the parasite, the absence of unsaturated fatty acids due to a low activity of desaturases was observed.