In order study patterns of local antibody responses following mucosal immunization of mice via different routes, a method for collection of secretions directly from mucosal surfaces was developed. Mice were immunized on days 0, 10, 17, and 24 by administration of cholera toxin into the oral cavity, stomach, colon-rectum, or vagina. At sacrifice on day 32, absorbent wicks were placed in the oral cavity and, via an applicator tube, into the vagina and distal colon-rectum and along the entire small intestine after flushing of luminal contents. Protein was quantitatively extracted from wicks, and specific anti-cholera toxin immunoglobulin A (IgA) and IgG were measured by enzyme-linked immunosorbent assay. Concentrations of specific IgA in secretions at various mucosal sites were dramatically influenced by the route of immunization. Oral immunization effectively induced IgA in saliva, and the intragastric route was optimal for induction of IgA in the small intestine. High levels of specific IgA appeared on the colonic-rectal mucosal surface only after rectal delivery of antigen. Oral, gastric, and rectal immunizations also produced distant responses in the vagina. Following vaginal immunization, however, neither local nor distant IgA responses were detected. These results suggest that vaccines intended for protection of colonic-rectal and vaginal mucosal surfaces might best be administered by the rectal route.