Pneumocystis carinii causes pneumonitis in immunodeficient individuals and is a prevalent opportunistic infection of patients with AIDS. This pathogen resides extracellularly in the hypophase lining the alveolar epithelium, which is highly enriched in lung surfactant lipids. Procedures yielding highly pure organism preparations that enable reliable biochemical analyses of organisms isolated from the lungs of infected laboratory animals have been developed. The results of the present study revealed that the fatty acid profiles of total lipids, the neutral lipid traction, and individual neutral lipid classes of lungs from normal and immunosuppressed rats as well as P. carinii were grossly similar, although some quantitative differences were detected. One qualitative exception found was the detection in P. carinii of the rare fatty acid cis-9,10-epoxy stearic acid, which was not detected in the lipids of rat lungs. The detection of this fatty acid in P. carinii may also have important taxonomic implications. Unlike phospholipids, many of the fatty acids of nonmembrane neutral lipids may be utilized by P. carinii for other cellular functions, such as stored reserves for energy production and precursors for organism-specific membrane lipids. The present study represents the first report of detailed fatty acid analyses of individual neutral lipid classes of this important opportunistic pathogen.