Food is an important vehicle for transmission of Shiga toxin-producing Escherichia coli (STEC). To assess the potential public health impact of STEC in Swiss raw milk cheese produced from cow's, goat's, and ewe's milk, 1,422 samples from semihard or hard cheese and 80 samples from soft cheese were examined for STEC, and isolated strains were further characterized. By PCR, STEC was detected after enrichment in 5.7% of the 1,502 raw milk cheese samples collected at the producer level. STEC-positive samples comprised 76 semihard, 8 soft, and 1 hard cheese. By colony hybridization, 29 STEC strains were isolated from 24 semihard and 5 soft cheeses. Thirteen of the 24 strains typeable with O antisera belonged to the serogroups O2, O22, and O91. More than half (58.6%) of the 29 strains belonged to O:H serotypes previously isolated from humans, and STEC O22:H8, O91:H10, O91:H21, and O174:H21 have also been identified as agents of hemolytic uremic syndrome. Typing of Shiga toxin genes showed that stx(1) was only found in 2 strains, whereas 27 strains carried genes encoding for the Stx(2) group, mainly stx(2) and stx(2vh-a/b). Production of Stx(2) and Stx(2vh-a/b) subtypes might be an indicator for a severe outcome in patients. Nine strains harbored hlyA (enterohemorrhagic E. coli hemolysin), whereas none tested positive for eae (intimin). Consequently, semihard and hard raw milk cheese may be a potential source of STEC, and a notable proportion of the isolated non-O157 STEC strains belonged to serotypes or harbored Shiga toxin gene variants associated with human infections.