Shiga toxin-producing Escherichia coli (STEC) cause non-bloody or bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The aim of the present study was to validate a multiplex PCR for the STEC diagnosis based on the detection of stx1, stx2 and rfbO157 genes. The multiplex PCR validation was carried out in two independent laboratories in a parallel way. Work range, selectivity and robustness were established. The PCR performance was evaluated using different concentrations of two STEC strains harboring different target genes. The work range depended on the strain analyzed, the maximum and the minimum values were 6.6 x 10(7) and 1.0 x 10(4) CFU/50 microl. The detection limit was 1.0 x 10(4) CFU/50 microl and the cut limit 1.0 x 10(5) CFU/50 ml. A good robustness was observed when different variables were introduced. Inclusivity, exclusivity, positive predictivity, negative predictivity and analytical accuracy were of 100%. Interference was not shown when different concentrations of STEC strains, carrying different genes, were used. The validated technique is an appropriate alternative for detection and confirmation of STEC O157 and non-O157 strains from bacterial cultures.