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To investigate the presence of diarrheagenic Escherichia coli in Lambaréné, Gabon, 150 children with diarrhea were screened for enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), and enteroinvasive E. coli (EIEC) using polymerase chain reaction and an HEp-2 cell culture techniques. Isolates of EAEC were detected in 57 children, two thirds of them between six months and two years of age, and isolates of ETEC were detected in seven patients. Isolates of EPEC, EHEC, and EIEC were not present in this population. Among the EAEC, the pCVD432 plasmid, a heat stable (ST)-like (ST-like) enterotoxin (EAST), and a plasmid-encoded heat-labile toxin (PET) were detected in 19, 34, and 42 cases, respectively. Detection of pCVD432, EAST, and PET were significantly associated with EAEC identified by the HEp-2 cell assay. Although detected only in 16 patients, the presence of the fimbriae AAF I (aagA) and AAF II (aafA) were more likely to occur in EAEC than in non-EAEC (odds ratio [OR] = 4.1, 95% confidence interval [CI] = 0.5–38.6, and OR = 2.3, 95% CI = 1.0–5.3, respectively). The EAEC isolates exhibited decreased susceptibility for ampicillin, tetracycline, and trimethoprim.