Aim To determine the occurrence and characteristics of Shiga toxin-producing Escherichia coli (STEC) in drinking water supplies treated and untreated. Methods and Results Drinking water samples (n=1850) were collected from 41 municipalities in the north of Parana State between February 2005 and January 2006. Escherichia coli isolates (n=300) were recovered from water and investigated for the presence of virulence markers related to STEC by PCR. STEC isolates recovered were then characterized for both phenotypic and genotypic traits. A total of 12 isolates (11 from untreated water and one from treated water) were positive for stx, including five positive for both stx1 and stx2, two positive for stx1 and five positive for stx2. None of the STEC isolates contained eae, but other virulence genes were observed such as ehxA (100%), saa (100%), lpfAO113 (75%), iha (42%), subAB (25%) and cdtV (8%). Multidrug resistance was identified in 25% of the STEC isolates. The 12 STEC isolates belonged to seven distinct serotypes and pulsed-field gel electrophoresis typing revealed the presence of two clusters and two clones in this region. Conclusion Drinking water, especially from untreated water supplies, can be source of STEC strains potentially pathogenic for humans. Significance and Impact of the Study The investigation of the drinking water supplies for pathogenic E.coli, as STEC, may be useful to prevent waterborne outbreaks.