Genetic variation among isolates of Escherichia coli O111 obtained mostly from patients with diarrhea in Brazil was assessed by multilocus enzyme electrophoresis to characterize chromosomal genotypes and by gene probes and adherence assays to characterize virulence properties. Among the 152 isolates, we resolved 16 distinct electrophoretic types (ETs), which differed on average at 40% of the enzyme loci. We identified four major bacterial O111 clones of different disease classes: ET 12, which includes the bulk of the enteropathogenic E. coli strains, typically showing localized adherence and intimate attachment in tissue culture assays; ET 1, which includes strains with a different set of virulence markers; ET 9, which includes strains that show intimate attachment but lack localized adherence and Shiga-like toxin genes; and ET 8, which includes strains that are Shiga-like toxin producers and have the corresponding traits of enterohemorrhagic E. coli. Enteroaggregative strains constituted ET 10 and also occurred in ET 1. Isolates of the major clones were found in South and North America and matched in ET and virulence factors to previously described diarrheagenic clones that are widely disseminated in the human population. Because the major clones are genetically distantly related and exhibit different combinations of virulence factors, we hypothesize that they have distinct mechanisms of pathogenesis. The results indicate that genetic divergence of bacteria with the O111 antigen, as measured by allelic variation in enzyme loci, is accompanied by divergence in virulence properties of clones so that identification and classification of pathogenic E. coli strains cannot be based solely on serotyping or a single virulence factor.