Cell-associated hemagglutinin-negative mutants were derived from cholera enterotoxin-negative Vibrio cholerae JBK70 by Tn5 mutagenesis. One of the mutants identified, SB001, was characterized in greater detail. Its ability to colonize ilea of adult rabbits was determined by feeding approximately 10(8) V. cholerae to each animal. At 17 h after feeding, the numbers of viable vibrios in the ilea were determined. There was a significant, 4 log, decrease in the ability of the hemagglutinin-negative mutant to colonize ileal tissue compared with the parent strain JBK70. In addition, the higher levels of colonization attained by JBK70 and the wild-type parent of JBK70, N16961, were associated with intestinal fluid accumulation and death. Rabbits immunized orally with approximately 10(8) SB001, when challenged 3 weeks later with either homologous biotype and serotype El Tor Inaba N16961 or heterologous Classical Ogawa 395, were protected to the same extent as those animals immunized with either the challenge strain or JBK70. This was evidenced by decreases in both the number of animals showing detectable colonization and the level of colonization achieved. A hemagglutinin-negative mutant of V. cholerae may therefore be of potential use as a live oral vaccine against cholera.