The therapy of Pneumocystis carinii (PC) pneumonia is often unsuccessful, particularly in patients with acquired immune deficiency syndrome (AIDS). Because of difficulties in growing the organism in vitro or obtaining purified organisms, current treatment choices have been made with little information on the metabolic effects of therapeutic agents on PC. This report quantitates the effects of the commonly used antifolates as well as the classic antineoplastic antifolate methotrexate and a lipid-soluble analogue, trimetrexate, on the target enzyme, dihydrofolate reductase (DHFR), in the PC organisms. Trimethoprim and pyrimethamine were found to be weak inhibitors (ID50 = 39,600 and 2,800 nM, respectively), while methotrexate and trimetrexate were potent reductase inhibitors (ID50 = 1.4 and 26.1 nM, respectively). transport studies with radiolabeled compounds showed that compounds with the classic folate structure (methotrexate and leucovorin) were not taken up by the intact PC organisms. In contrast, trimetrexate exhibited rapid uptake. These results suggest a major therapeutic advantage may be gained by combining a potent, readily transported PC DHFR inhibitor such as trimetrexate with the reduced folate leucovorin to achieve a highly potent antiprotozoan effect while preventing toxicity to mammalian cells.