The resistance of Escherichia coli O157:H7 to amoxicillin/clavulanic acid, ampicillin, ceftazidime, ceftriaxone, cefuroxime, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, streptomycin, sulfisoxazole, tetracycline, ticarcillin, tobramycin, and trimethoprim-sulfamethoxazole was examined, and resistant strains were characterized. All 56 isolates collected between 1984 and 1987 were susceptible to all antibiotics tested; 13 (7.4%) of 176 strains isolated between 1989 and 1991 were resistant to streptomycin, sulfisoxazole, and tetracycline. lambda-restriction fragment length polymorphism analysis suggested that the 13 resistant strains belonged to nine different clones. The emerging resistance of E. coli O157:H7 to antibiotics could portend an increased prevalence of this pathogen in food animals that receive antibiotics. Antimicrobial resistance of E. coli O157:H7 could be useful as a rapid epidemiologic marker and as a way to select this pathogen from suspected vehicles of transmission, but this resistance could also complicate therapeutic trials with sulfa-containing antibiotics.