In Mellassine (a major city in the state of Tunis) and Ben Arous state (south east of Tunis), a total of 212 stool samples were collected from children and adults (symptomatic and asymptomatic groups) between November 2001 and November 2004. Three hundred and twenty-seven E. coli strains were isolated and studied, to look for shiga toxin-producing Escherichia coli (STEC) strains, which were further analysed to investigate and determine clonal relationship among Tunisian STEC strains isolated from different sources (diarrheal cases and food products). They were analysed to characterize their serotypes, virulence genes by PCR, cytotoxic effect on Vero cell, plasmid profiles, and pulsed-field gel electrophoresis (PFGE) patterns. Eleven isolates (10 nontypeable, one O157:H7) carried stx gene and shared Stx restriction fragment length polymorphism (RFLP) patterns (stx1 (+) , stx2 (+) ). Seven of these strains were isolated from acute diarrheal cases, and four were isolated from a control group (among which the only isolated STEC O157:H7). Two of the STEC strains harboured both eae and ehxA genes. Analysis of the cytotoxic effect on Vero cells showed that a correlation exists between carrying stx1 (+) , stx2 (+) genes and cytotoxicity. Also a correlation was noticed between STEC strains recovered from different sources regarding plasmid profiles and PFGE patterns. All stool samples positive for STEC were nonbloody. None of the STEC-positive patients developed severe diseases. These data demonstrate that although STEC is not a major cause of acute diarrhea in Tunis, it should not be overlooked. Measures should be taken to improve the detection and isolation of STEC from acute diarrheal cases as well as carriers.