Vibrio cholerae EI Tor strains from Peru, Bangladesh, and Bahrain were attenuated by deletion of a genetic element that encodes virulence factors and RSI. The B subunit of ctx (ctxB) was reintroduced into the recA gene of the deletion mutants, rendering them unable to recombine with exogenous genetic elements and generating Peru-3, Bang-3, and Bah-3. Fifteen volunteers received one dose of various vaccine strains at 4 × 106 to 1 × 108 cfu. All strains colonized the gut. A ≥ 4-fold rise in vibriocidal titer was observed in 14 volunteers, with titers of ≥ 1600 in 13. Peru-3 was the least reactogenic, but 2 of 6 volunteers had loose stools. Peru-14, a filamentous motility-deficient mutant of Peru-J, was well tolerated and colonized 18 of 21 volunteers at doses of 2 × 106 to 1 × 109 cfu. Also, when 8 Peru-3 or Peru-S vaccinees, 5 Peru-14 vaccinees, and 8 controls were challenged with 2 × 106 cfu V. cholerae EI Tor Inaba (NI6961), 11 vaccinees were protected compared with no controls. Peru-14 shows promise as a safe, effective, single-dose oral vaccine against EI Tor cholera.