Iclaprim, a novel dihydrofolate reductase inhibitor, inhibited 90% of the clinical isolates (MIC(90)) of Streptococcus pneumoniae (n = 785) collected by a national surveillance program at a concentration of 1 microg/ml. The MIC(90) for iclaprim was 7 doubling dilutions lower for trimethoprim-sulfamethoxazole-susceptible isolates (n = 670; MIC(90), 0.06 microg/ml) than for trimethoprim-sulfamethoxazole-resistant isolates (n = 115; MIC(90), >or=8 microg/ml). The potential clinical utility of iclaprim to treat patients with pneumococcal infections may depend upon the current prevalence of resistance to trimethoprim-sulfamethoxazole in this pathogen.