The pulsed-field gel electrophoresis (PFGE) patterns of 46 Shiga toxin-producing Escherichia coli (STEC) strains isolated in São Paulo, Brazil, during the period from 1976 to 2003 were compared with those found among 30 non-STEC strains that carried eae and that belonged to the same serogroups as the STEC strains. All except two of the STEC and non-STEC strains of human origin were from sporadic and unrelated cases of infection; two O111 strains originated from the same patient. Multiple PFGE patterns were found among STEC strains of distinct serotypes. Moreover, the PFGE restriction patterns of STEC strains differed substantially from those observed among non-STEC strains of the same serogroup except serotype O26 strains. Based on the indistinguishable PFGE pattern for two O157:H7 STEC strains isolated in the same geographic area at an interval of approximately 15 days and toxin profile data, the first occurrence of an O157:H7 outbreak in Brazil during that period can be suggested. In general, a close relationship between types of intimin, serotypes, and diarrheagenic groups of E. coli was observed. This is the first time that a large collection of STEC strains from Brazil has been analyzed, and a great genetic diversity was shown among O157:H7 and non-O157:H7 STEC strains isolated in São Paulo, Brazil.