Several drugs that interact with membrane sterols or inhibit their syntheses are effective in clearing a number of fungal infections. The AIDS-associated lung infection caused by Pneumocystis jirovecii is not cleared by many of these therapies. Pneumocystis normally synthesizes distinct C₂₈ and C₂₉ 24-alkylsterols, but ergosterol, the major fungal sterol, is not among them. Two distinct sterol compositional phenotypes were previously observed in P. jirovecii. One was characterized by Δ⁷ C₂₈ and C₂₉ 24-alkylsterols with only low proportions of higher molecular mass components. In contrast, the other type was dominated by high C₃₁ and C₃₂ 24-alkylsterols, especially pneumocysterol. In the present study, 28 molecular species were elucidated by nuclear magnetic resonance analysis of a human lung specimen containing P. jirovecii representing the latter sterol profile phenotype. Fifteen of the 28 had the methyl group at C-14 of the sterol nucleus and these represented 96% of the total sterol mass in the specimen (excluding cholesterol). These results strongly suggest that sterol 14α-demethylase was blocked in these organisms. Twenty-four of the 28 were 24-alkylsterols, indicating that methylation of the C-24 position of the sterol side chain by S-adenosyl-l-methionine:sterol C-24 methyl transferase was fully functional.