This study aims to assess the prevalence of Shiga toxin-producing Escherichia coli (STEC) in ground beef collected in two cities located in the State of São Paulo, Brazil. A total of 250 samples of raw ground beef were collected in local grocery stores during the period of March to December 2002 in the cities of Ribeirão Preto (114 samples) and Campinas (136 samples), São Paulo State, Brazil. The samples were processed according to standard methods. The resulting 591 E.coli colonies were screened for STEC by hybridization assays using the specific DNA probes, stx1, stx2 and eae. Further characterization of STEC isolates included the search for the ehxA sequence, detection of enterohemolysin and expression of Shiga toxin using the Vero cell assay. STEC isolates belonging to serotypes O93:H19, ONT:HNT, ONT:H7, and O174:HNT we recovered from four samples (3.5%) collected in Ribeirão Preto. All samples from Campinas were negative for STEC. Three of the strains carried stx2 and ehxA sequences while one harbored stx1, stx2 and ehxA sequences. Considering that among foods of animal origin, ground beef is an important vehicle for STEC transmission, these data emphasize the need of a closer surveillance of these microorganisms. They can survive in unfavorable conditions specially when the products are refrigerated or frozen for long periods of time and can be the cause of outbreaks affecting a great number of consumers.