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The antimicrobial susceptibility of 50 Clostridium difficile isolates, 36 of them from horse feces and 14 from environmental sites, was determined by broth microdilution. The antimicrobial agents tested were avilamycin, cephalothin, chloramphenicol, clindamycin, erythromycin, gentamicin, neomycin, oxacillin, oxytetracycline, penicillin, spiramycin, streptomycin, trimethoprim/sulfamethoxazole, vancomycin, and virginiamycin. All isolates were susceptible to vancomycin (MIC/=1 microg/ml). The MICs of erythromycin, oxytetracycline, spiramycin, and virginiamycin showed a bimodal distribution. Compared with the majority of isolates, the MICs of erythromycin (MIC16 microg/ml), oxytetracycline (MIC/=32 microg/ml), spiramycin (MIC16 microg/ml), and virginiamycin (MIC 8-16 microg/ml) were higher for 18 isolates. Those were mainly isolated from horses at animal hospitals and further from environmental sites at a stud farm. In contrast, all isolates, except one, from healthy foals had low MICs of erythromycin, spiramycin, virginiamycin, and oxytetracycline. The isolates from soil in public parks had also low MICs of these antimicrobial agents. Broth microdilution appeared both reliable and reproducible for susceptibility testing of C. difficile. The method was also readily performed and the MIC endpoints were easily read.