We previously identified two subtypes of the epidemic strain Clostridium difficile PCR ribotype 1, one clindamycin-sensitive strain (arbitrarily primed PCR [AP-PCR] type Ia) and a closely related clindamycin-resistant strain (AP-PCR type Ib) in our institution. We have now carried out prospective epidemiological surveillance for 4 years, immediately following the relocation of two acute medicine wards for elderly patients (wards A and B), to determine the clinical epidemiology of subtypes of the epidemic C. difficile PCR ribotype 1 group. To maximize the chance of strain discrimination, we used three DNA fingerprinting methods, AP-PCR, ribospacer PCR (RS-PCR), and pulsed-field gel electrophoresis (PFGE), to analyze C. difficile isolates recovered from symptomatic patients and from repeated environmental samplings. On ward B the incidence of C. difficile infection correlated significantly with the prevalence of environmental C. difficile both in ward areas closely associated with patients and health care personnel (r = 0.53; P < 0.05) and in high-reach sites (r = 0.85; P < 0.05). No such relationships were found on ward A. Seventeen distinct C. difficile genotypes were identified, 17 by AP-PCR, 12 by PFGE, and 11 by RS-PCR, but only 4 of 17 genotypes caused patient infection. Isolates recovered from the hospital ward environment were much more diverse (14 genotypes). AP-PCR type Ia represented >90% of the C. difficile isolates. In addition to this genotype, only two others were isolated from both patient feces and environmental surfaces. AP-PCR type Ib (clindamycin-resistant PCR ribotype 1 clone) was not associated with any cases of C. difficile infection and was isolated from the environment on only two occasions, after having been implicated in a cluster of six C. difficile infections 5 months before this study. The disappearance of this strain implies that differences in virulence and/or selective pressures may exist for this strain and the closely related, widespread C. difficile AP-PCR type Ia strain. Our findings emphasize the need to understand the epidemiology and virulence of clinically significant strains to determine successful control measures for C. difficile infections.