We compared Ro 11-8958, an analog of trimethoprim (TMP) with improved antimicrobial and pharmacokinetic properties, other dihydrofolate reductase (DHFR) inhibitors, sulfamethoxazole (SMX), and dapsone (DAP) in the treatment of Pneumocystis carinii pneumonia in an immunosuppressed rat model. In contrast to previous reports, high dosages of the DHFR inhibitors were used in combination with fixed, low dosages of SMX (3 mg/kg of body weight per day) or DAP (25 mg/kg/day). When administered alone at these dosages, SMX and DAP reduced the median P. carinii cyst count about 5- to 15-fold. Ro 11-8958, TMP, and diaveridine used at a dosage of 20 mg/kg/day with SMX were only slightly more effective than SMX used alone. However, administration of these DHFR inhibitors at a dosage of 100 mg/kg/day with SMX lowered the cyst count about 500- to 1,000-fold, indicating a synergistic effect. Little or no synergism was found when other DHFR inhibitors (pyrimethamine, cycloguanil, and tetroxoprim) were combined with SMX. Regimens of Ro 11-8958 at a dosage of 20 mg/kg/day with DAP and of TMP or diaveridine used at a dosage of 100 mg/kg/day with DAP showed comparable anti-P. carinii activity, lowering the cyst count 100- to 200-fold. By contrast, Ro 11-8958 administered at a dosage of 100 mg/kg/day with DAP reduced the cyst count > 1,000-fold. Thus, the experimental approach used here enables the rat model of pneumocystosis to be used to compare synergistic combinations of antifolate drugs. The favorable results achieved with Ro 11-8958 indicate that it should be considered for clinical trials.