Fecal swabs obtained from 1,300 healthy lambs in 93 flocks in Spain in 1997 were examined for Shiga toxin-producing Escherichia coli (STEC). STEC O157:H7 strains were isolated from 5 (0.4%) animals in 4 flocks, and non-O157 STEC strains were isolated from 462 (36%) lambs in 63 flocks. A total of 384 ovine STEC strains were characterized in this study. PCR showed that 213 (55%) strains carried the stx(1) gene, 10 (3%) possessed the stx(2) gene, and 161 (42%) carried both the stx(1) and the stx(2) genes. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 106 (28%) and 23 (6%) of the STEC strains, respectively. The STEC strains belonged to 35 O serogroups and 64 O:H serotypes (including 18 new serotypes). However, 72% were of 1 of the following 12 serotypes: O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O136:H20, O146:H8, O146:H21, O156:H-, O166:H28, and ONT:H21 (where NT is nontypeable). Although the 384 STEC strains belonged to 95 different seropathotypes (associations between serotypes and virulence genes), 49% of strains belonged to only 11. O91:H- stx(1) stx(2) (54 strains) was the most common seropathotype, followed by O128:H- stx(1) stx(2) (33 strains) and O6:H10 stx(1) (25 strains). Three strains of serotypes O26:H11, O156:H11, and OX177:H11 had intimin type beta1; 5 strains of serotype O157:H7 possessed intimin type gamma1; and 15 strains of serotypes O49:H-, O52:H12, O156:H- (12 strains), and O156:H25 had the new intimin, intimin type zeta. The majority (82%) of ovine STEC strains belonged to serotypes previously found to be associated with human STEC strains, and 51% belonged to serotypes associated with STEC strains isolated from patients with hemolytic-uremic syndrome. Thus, this study confirms that healthy sheep are a major reservoir of STEC strains pathogenic for humans.