Unlike Shiga toxin 2 (stx2) genes, most nucleotide sequences of Shiga toxin 1 (stx1) genes from Shiga toxin-producing Escherichia coli (STEC), Shigella dysenteriae, and several bacteriophages (H19B, 933J, and H30) are highly conserved. Consequently, there has been little incentive to investigate variants of stx1 among STEC isolates derived from human or animal sources. However stx1OX3, originally identified in an OX3:H8 isolate from a healthy sheep in Germany, differs from other stx1 subtypes by 43 nucleotides, resulting in changes to 12 amino acid residues, and has been renamed stx1c. In this study we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that distinguishes stx1c from other stx1 subtypes. The PCR-RFLP assay was used to study 378 stx1-containing STEC isolates. Of these, 207 were isolated from sheep, 104 from cattle, 45 from humans, 11 from meat, 5 from swine, 5 from unknown sources, and 1 from a cattle water trough. Three hundred fifty-five of the 378 isolates (93.9%) also possessed at least one other associated virulence gene (ehxA, eaeA, and/or stx2); the combination stx1, stx2, and ehxA was the most common (175 of 355 [49.3%]), and 90 of 355 (25.4%) isolates possessed eaeA. One hundred thirty-six of 207 (65.7%) ovine isolates possessed stx1c alone and belonged to 41 serotypes. Seventy-one of 136 (52.2%) comprised the common ovine serotypes O5:H−, O128:H2, and O123:H−. Fifty-two of 207 isolates (25.1%) possessed an stx1 subtype; 27 (51.9%) of these belonged to serotype O91:H−. Nineteen of 207 isolates (9.2%) contained both stx1c and stx1 subtypes, and 14 belonged to serotype O75:H8. In marked contrast, 97 of 104 (93.3%) bovine isolates comprising 44 serotypes possessed an stx1 subtype, 6 isolates possessed stx1c, and the remaining isolate possessed both stx1c and stx1 subtypes. Ten of 11 (91%) isolates cultured from meat in New Zealand possessed stx1c (serotypes O5:H−, O75:H8/H40, O81:H26, O88:H25, O104:H−/H7, O123:H−/H10, and O128:H2); most of these serotypes are commonly recovered from the feces of healthy sheep. Serotypes containing stx1 recovered from cattle rarely were the same as those isolated from sheep. Although an stx1c subtype was never associated with the typical enterohemorrhagic E. coli serogroups O26, O103, O111, O113, and O157, 13 human isolates possessed stx1c. Of these, six isolates with serotype O128:H2 (from patients with diarrhea), four O5:H− isolates (from patients with hemolytic-uremic syndrome), and three isolates with serotypes O123:H− (diarrhea), OX3:H8 (hemolytic-uremic syndrome), and O81:H6 (unknown health status) represent serotypes that are commonly isolated from sheep.