Studies in the infant mouse cholera model have evaluated the significance of toxin-coregulated pili (TCP) in the pathogenesis of Vibrio cholerae strains of El Tor biotype. Four El Tor strains--two which produce TCP during in vitro growth and two which do not--were mutated by the insertion of an antibiotic-resistance cartridge into the tcpA gene (encoding the pilin monomer). The resulting mutants were otherwise indistinguishable from wild-type and in particular were unaltered in their sensitivity to antibody-dependent, complement-mediated bacteriolysis. All were dramatically attenuated and showed a marked impairment in terms of in vivo persistence in mixed-infection competition experiments. Virulence was restored by provision of a functional tcp operon in trans, confirming that the pathogenic potential of El Tor strains is critically dependent upon product(s) of this operon.