The distribution of the stx(1c) allele among Shiga toxin (Stx)-producing Escherichia coli (STEC) and the virulence characteristics of stx(1c)-harboring STEC are unknown. In this study, we identified stx(1c) in 76 (54.3%) of 140 eae-negative, but in none of 155 eae-positive, human STEC isolates (P < 0.000001). The 76 stx(1c)-harboring E. coli isolates belonged to 22 serotypes, and each produced Stx1c as demonstrated by latex agglutination. Characterization of putative virulence factors demonstrated the presence of the locus of proteolysis activity (LPA) and the high-pathogenicity island in 65.8 and 21.1%, respectively, of the 76 Stx1c-producing E. coli isolates. Moreover, all but three of these strains contained saa, the gene encoding an STEC autoagglutinating adhesin. The virulence profiles of Stx1c-producing E. coli isolates were mostly serotype independent and heterogeneous. This enabled us to subtype the isolates within the same serotype. The individuals infected with Stx1c-producing E. coli strains were between 3 months and 72 years old (median age, 23.5 years) and usually had uncomplicated diarrhea or were asymptomatic. We conclude that Stx1c-producing E. coli strains represent a significant subset of eae-negative human STEC isolates, which belong to various serotypes and frequently possess LPA and saa as their putative virulence factors. The phenotypic and molecular characteristics determined in this study allow the subtyping of Stx1c-producing STEC in epidemiological and clinical studies.