Pneumocystis carinii synthesizes folates de novo from exogenous p-aminobenzoic acid (pABA). Lung-derived organisms take up [3H]pABA in vitro except in the presence of sulfamethoxazole. Supernatants from spinner-flask cultures take up [3H]pABA if they were inoculated with lungs from infected rats, but not if they were inoculated with lungs from uninfected rats. P. carinii folates consist primarily of pteroylpentaglutamates. Plasmodium falciparum, in contrast, contains primarily pteroyltetraglutamates. Culture-derived organisms synthesize folates at a four-fold higher specific activity than lung-derived organisms, possibly because they contain less contaminating lung debris. These data suggest that P. carinii remains metabolically active in culture for at least 4 days.