Molecular techniques were used for studying the epidemiology of diarrhoeal infections due to Escherichia coli in the Gauteng region in South Africa. In total, 151 E. coli strains isolated from stools of patients with diarrhoea and 30 strains isolated from stools of healthy individuals were collected between March 1996 and May 1997. The E. coli isolates were characterized by serotyping, antimicrobial susceptibility testing, and adherence patterns. Polymerase chain reaction (PCR) was performed to determine the presence of the genes-encoding virulence factors. PCR showed that 59 (32.6%) of the E. coli isolates carried eaeA genes, 6 (3.3%) possessed bfpA genes, 4 (2.2%) CNF1, and 2 (1.1%) carried labile toxin and Stx2 genes. The eae genes were more prevalent in strains isolated from patients than in those from the control group (p < 0.001). Forty-eight (26.5%) strains belonged to enteropathogenic E. coli (EPEC) O serogroups and 14 (7.7%) to Shiga toxin-producing E. coli (STEC) O157 serotype. A high percentage (28.2%) of atypical EPEC strains possessing the eaeA but not the bfpA genes was isolated. Most isolates were susceptible to commonly-used antimicrobial agents. The adherence of the E. coli strains to HeLa cells was identified more in patients (69.4%) than in the control group (60%) and was more dominant in infants than in adults. PCR and tissue culture assays were shown to be useful techniques for the epidemiological study of E. coli where this organism is a major cause of diarrhoea.