Enterotoxigenic Escherichia coli (ETEC) strains remain a formidable cause of diarrheal disease. To identify novel surface proteins of ETEC, we performed TnphoA mutagenesis of prototype ETEC strain H10407 and discovered a secreted protein not previously recognized in ETEC. DNA sequencing of the interrupted locus in mutant TnphoA.977 revealed a candidate 4,095-bp open reading frame without significant homology to commensal E. coli K-12 genomic DNA. Translation of this sequence revealed that it encoded a predicted peptide of 147.7 kDa that bears significant homology to members of the autotransporter family of bacterial virulence factors, particularly the serine protease autotransporters of the Enterobacteriaceae proteins. The gene identified in H10407, eatA (ETEC autotransporter A), encodes a potential serine protease motif (GDSGSP) in the secreted amino-terminal domain, and the predicted peptide shows more than 80% homology with SepA, a virulence protein secreted by Shigella flexneri. DNA hybridization and PCR demonstrated that eatA resides on the 92-kDa pCS1 virulence plasmid of H10407 and that it is present in multiple clinical ETEC strains. Immunoblots with antisera directed against a recombinant EatA passenger protein fragment identified a 110-kDa protein in supernatants purified from H10407 but not from the TnphoA.977 mutant or H10407-P, which lacks pCS1. EatA possesses serine protease activity that is abolished by mutations within a serine protease catalytic triad formed by residues H(134), D(162), and S(267). Finally, interruption of the eatA gene retarded fluid accumulation in the rabbit ileal loop model, suggesting that this autotransporter contributes to the virulence of ETEC.