The in vitro interaction of trimethoprim and sulfamethoxazole on clinical isolates of Paracoccidioides brasiliensis was studied. With complete inhibition and a visual endpoint used as the criteria, three of four strains had minimal inhibitory concentrations that indicated resistance to sulfamethoxazole, and all four strains were resistant to trimethoprim. A marked synergism in inhibition was noted with the combination of these drugs against sulfa-resistant strains. A sulfamethoxazole/trimethoprim ratio of 5:1 was the most synergistic. Fifty percent inhibition, determined spectrophotometrically, of the strains could be achieved with sulfamethoxazole alone. In summary, the striking synergy observed suggests that combination chemotherapy with these drugs deserves further study.