BACKGROUND We characterized 36 atypical enteropathogenic Escherichia coli (EPEC) serotypes isolated from children with and without diarrhea in Iran. Because the identification of atypical EPEC based on biochemical features is rather difficult and time consuming, we used a combination of three approaches, including a polymerase chain reaction-based method, culture adherence assay, and the restriction analysis of fliC gene (fliC-restriction fragment length polymorphism), to identify E coli serotypes. METHODS To distinguish typical and atypical EPEC strains, the presence of EPEC attaching effacing A gene (eaeA) gene and EPEC-attaching factor (EAF) plasmid were analyzed. All E coli strains were identified based on the detection of the eaeA(+), bundle-forming pili A gene (bfpA(-)), EAF(-) or eaeA(-), bfpA(+), EAF(-) profiles and the absence of stx (encoded for shiga toxin) gene as atypical EPEC. RESULTS All strains studied belonged to 5 atypical EPEC serogroups and 15 serotypes based on the virulence profiles. Of 36 atypical EPEC serotypes, 22 (61.2%) and 14 (38.8%) strains isolated from diarrheal and healthy cases, respectively. O142:H48 (19.5%) and O111:H21 (11.1%) serotypes were the most prevalent isolates, followed by serotypes O111: H(-) and O86:H48 (5.6% each). CONCLUSIONS The characteristics of the atypical EPEC serotypes from children with diarrhea were significantly different from those without diarrhea. The compilation of data on atypical EPEC strains presented here indicates the importance of a combined approach of conventional and molecular tests to study the virulence and epidemiology of EPEC serotypes in human subjects.