Trimethoprim and sulfamethoxazole were tested alone and in combination against 227 recently isolated Shigella strains. Variations in medium constituents and inoculum size were used to determine the optimal testing conditions. The plate dilution method with addition of 5% lysed horse blood to the susceptibility test medium and an inoculum size of 10(2) organisms was found to provide satisfactory results. All 227 strains were inhibited by low concentrations of trimethoprim, and all were susceptible to the combination of 0.06 mug of trimethoprim per ml and 1.25 mug of sulfamethoxazole per ml. Sixteen percent of these strains were resistant to ampicillin, 33% to tetracycline, 15% to chloramphenicol, and 27% to cephalothin. Based on these in vitro observations, trimethoprim and sulfamethoxazole appear worth evaluating for treatment of shigellosis due to multiply antibiotic-resistant strains.