Spontaneous and chemically induced mutants with reduced ability to produce cholera enterotoxin (choleragen) as an extracellular protein were isolated from Vibrio cholerae strains 569B Inaba, a classical cholera vibrio, and 3083-2 Ogawa, an El Tor vibrio. By qualitative and quantitative immunological assay in vitro such mutants could be separated into different classes characterized either by production of no detectable choleragen (tox minus), or of small quantities of extracellular choleragen, or of large quantities of cell-associated choleragen but little extracellular choleragen. Analysis of proteins in concentrated culture supernates by electrophoresis in polyacrylamide gels showed that cultures from tox minus strains lacked proteins with electrophoretic mobilities corresponding with choleragen or the spontaneously formed toxoid (choleragenoid). Infant rabbits infected with the tox minus strains remained asymptomatic or developed milder symptoms than rabbits infected with the tox+ parental strains. When symptoms of cholera developed after inoculation with tox minus mutants, detectable numbers of tox+ revertants could be isolated from the intestines of the infected animals. Two tox minus strains, designated M13 and M27, caused no sumptoms and showed no evidence of reversion to tox+ during single passage in infant rabbits, and mutant M13 also remained avirulent and stably tox minus during six cycles of serial passage in infant rabbits. Strains M13 and M27 were also noncholeragenic in acult rabbit ileal loops. Quantitative cultures of the intestines from infected infant rabbits demonstrated that the avirulent mutant M13 can multiply in vivo and can persist in the intestinal tract for at least 48 h.