This study investigated the applicability of molecular epidemiological techniques to the identification of the causal agent of an outbreak of diarrhea caused by ingestion of food contaminated with enterotoxigenic Escherichia coli (ETEC). The outbreak occurred at four elementary schools in July 1996 and affected more than 800 people. Illness was most strongly associated with eating tuna paste (relative risk, 1.79; 95% confidence interval = 1.16 to 2.79; P = 0.0001). To evaluate the epidemiological characteristics of the pathogen, the DNAs from numerous isolated ETEC strains were subjected to randomly amplified polymorphic DNA analysis, pulsed-field gel electrophoresis of nuclease S1-treated plasmid DNA, and analysis of genomic DNA restriction fragment length polymorphisms. All ETEC isolates were of the O25:NM (nonmotile) serotype, which carries a heat-stable enterotoxin Ib gene. Genotypic analysis demonstrated that the strains isolated from the patients at all four schools were identical. The isolates of ETEC O25:NM obtained from the tuna paste that had been served for lunch at these schools were genetically indistinguishable from those isolated from the patients. Results suggest that this outbreak was food borne. The molecular biology-based epidemiological techniques used in this study were useful in characterizing the causal agent in this food-borne epidemic.