The sterol composition of Pneumocystis carinii, an opportunistic pathogen responsible for life-threatening pneumonia in immunocompromised patients, was determined. Our purpose was to identify pathway-specific enzymes to impair using sterol biosynthesis inhibitors. Prior to this study, cholesterol 15 (ca. 80% of total sterols), lanosterol 1, and several phytosterols common to plants (sitosterol 31, 24alpha-ethyl and campesterol, 24alpha-methyl 30) were demonstrated in the fungus. In this investigation, we isolated all the previous sterols and many new compounds from P. carinii by culturing the microorganism in steroid-immunosuppressed rats. Thirty-one sterols were identified from the fungus (total sterol = 100 fg/cell), and seven sterols were identified from rat chow. Unusual sterols in the fungus not present in the diet included, 24(28)-methylenelanosterol 2; 24(28)E-ethylidene lanosterol 3; 24(28)Z-ethylidene lanosterol 4; 24beta-ethyllanosta-25(27)-dienol 5; 24beta-ethylcholest-7-enol 6; 24beta-ethylcholesterol 7; 24beta,-ethylcholesta-5,25(27)-dienol 8; 24-methyllanosta-7-enol 9; 24-methyldesmosterol 10; 24(28)-methylenecholest-7-enol 11; 24beta-methylcholest-7-enol 12; and 24beta-methylcholesterol 13. The structural relationships of the 24-alkyl groups in the sterol side chain were demonstrated chromatographically relative to authentic specimens, by MS and high-resolution 1H NMR. The hypothetical order of these compounds poses multiple phytosterol pathways that diverge from a common intermediate to generate 24beta-methyl sterols: route 1, 1 --> 2 --> 11 --> 12 --> 13; route 2, 1 --> 2 --> 9 --> 10 --> 13; or 24beta-ethyl sterols: route 3, 1 --> 2 --> 4 --> 6 --> 7; route 4, 1 --> 2 --> 5 --> 8 --> 7. Formation of 3 is considered to form an interrupted sterol pathway. Taken together, operation of distinct sterol methyl transferase (SMT) pathways that generate 24beta-alkyl sterols in P. carinii with no counterpart in human biochemistry suggests a close taxonomic affinity with fungi and provides a basis for mechanism-based inactivation of SMT enzyme to treat Pneumocystis pneumonia.