Rats were parenterally immunized with bovine serum albumin (BSA) and horseradish peroxidase (HRP) emulsified in complete Freund's adjuvant; absorption of these antigens was tested in vitro by means of the everted gut sac technique. A significant and specific decrease in uptake of BSA was noted in rats injected five times with BSA, but not in rats injected only twice. Absorption of 125I-HRP and HRP, measured enzymatically, was also significantly inhibited in rats immunized five times with HRP. The binding of 125I-BSA to intestinal secretions, mucosal extracts and serum from rats showing interference with BSA absorption (multiple injections) was 2- to 10-fold greater than that of comparable preparations from rats which did not show interference (injected twice). Antigen-binding to IgGa, IgG1, and IgM antibodies was demonstrated by radioimmunoelectrophoresis in the serum of rats with multiple injections of BSA; in addition, binding to IgGa and IgG1 antibodies was demonstrated with mucosal extracts. Although antigen uptake was inhibited in rats immunized parenterally with BSA and HRP, the number of gut sacs showing an inhibited response and the extent of the response were less than that previously observed with gut sacs obtained from rats immunized orally with the same antigens. Binding activity of 125I-BSA in mucosal extracts from this latter group of rats was the same as in rats parenterally immunized but only IgG1 antibodies were noted in mucosal extracts. It was suggested that the more consistent inhibition of antigen uptake in orally immunized vs parenterally immunized rats may be related to the type or pattern of local antibody distribution.