We describe here a new animal model that offers the prospect of using conventional adult mice for direct evaluation of the protective potential of new cholera vaccines. Pretreatment of adult mice with oral streptomycin allowed intestinal colonization by streptomycin-resistant Vibrio cholerae strains of either the O1 or the O139 serogroup. Bacteria were recovered in greatest numbers from the cecum and large intestine, but recoveries from all regions of the gut correlated significantly with bacterial excretion in fresh fecal pellets, which thus provides a convenient indicator of the extent and duration of gut colonization. Mice immunized mucosally or systemically with viable or inactivated V. cholerae were shown to be comparatively refractory to colonization after challenge with the immunizing strain. Several variables were examined to optimize the model, the most significant being the size of the challenge inoculum; surprisingly, a smaller challenge dose resulted in more consistent and sustained colonization. Studies with mutant strains unable to produce cholera toxin or toxin-coregulated pili revealed that neither factor contributed significantly to colonization potential. Protection against V. cholerae challenge was shown to be serogroup restricted, and significant inverse correlations were detected between serum and intestinal anti-lipopolysaccharide antibody responses and the levels of excretion of challenge organisms.