Escherichia coli serogroup O111 is among the six most commonly reported non-O157:H7 Shiga toxin-producing E. coli (STEC), which are emerging as important foodborne pathogens. We have assembled a collection of environmental and clinical strains of E. coli O111 from diverse sources and investigated various genotypic and phenotypic characteristics of these strains to gain a better understanding of the epidemiology and biology of this serogroup. Sixty-three percent of the strains (24/38) were of H-type 8, which dominated the environmental- and outbreak-strains group, whereas the sporadic-case strains were more heterogeneous in H-type. All of the environmental and outbreak strains harbored the Shiga toxin 1 gene (stx1), eae, and ehx, and a subset of these also carried the Shiga toxin 2 gene (stx2). Only 9 of 16 sporadic-case strains produced stx1 and/or stx2, and these were mostly of H-type 8 and 10. Pulsed-field gel electrophoresis analysis revealed a cluster of environmental, outbreak, and sporadic illness strains with high phylogenetic similarity. Strains in this pulsogroup were all of the H8 type and STEC pathotype, and carried eae and ehx. Smaller clusters of highly similar STEC O111 strains included outbreak and sporadic illness strains isolated during different time periods or from different geographical locations. A distinct aggregative behavior was observed in the cultures of all environmental and outbreak STEC O111 strains, but not in those of sporadic-case strains. Among environmental and outbreaks strains, aggregation was positively correlated with production of curli fimbriae and RpoS function, and negatively with cellulose synthesis, while the nonaggregative behavior of sporadic-case strains correlated (positively) only with cellulose production. Our results indicate that STEC O111 strains sharing high genotypic similarity and important phenotypic traits with STEC O111 outbreak strains are present in the agricultural environment and may contribute to the burden of foodborne disease.