We developed and evaluated a method to genotypically identify enterotoxigenic Escherichia coli (ETEC) and to characterize these organisms with respect to 18 of 21 known colonization factors (CFs). The method, which is based on polynucleotide DNA-DNA colony hybridization, includes a pooled toxin probe assay to identify ETEC, and individual probe assays to detect the enterotoxins STp, STh, and LT, and the CFs CFA/I, CS1-CS8, CS12-CS15, CS17-CS19, CS21, and CS22. We evaluated the pooled toxin probe assay during a cohort study of childhood diarrhea, and the individual probe assays against 33 reference strains and 92 clinical ETEC isolates. There was close to a complete agreement between the pooled toxin probe assay and the individual toxin probe assays, and between the individual CF probe assays and the corresponding phenotypic assays.