Oral administration of protein antigen in solution routinely leads to development of oral tolerance in most mammals but has been reported to be fully immunogenic in chickens. Previous studies, including several performed by our laboratory, have demonstrated that oral administration of discrete amounts of BSA for 6 consecutive days is fully immunogenic. This study was performed to determine immunoresponsiveness to protein antigen administered ad libitum at low levels in drinking water compared with i.p. and oral gavage routes of administration. Seven days following the last oral immunization, serum was assayed for IgG, bile for IgA, and tissue culture supernatant from 3 distinct lower intestinal regions for IgG and IgA in immunized and nonimmunized single-comb White Leghorn chickens. Systemic responses in the serum of experimental birds revealed a greater (P < 0.001) IgG response when BSA was administered via i.p. injection or by drinking water compared with gavage administration or nonimmunized controls. Responses measured in bile revealed that BSA administration in the drinking water resulted in a greater (P < 0.001) secretory IgA response compared with i.p. or gavage administration, and negative control groups. Intestinal antigen specific IgG, but not IgA, was elevated (P < 0.05) in all intestinal areas tested in birds immunized against BSA by drinking water and i.p. routes of administration, compared with other experimental groups. Taken together, the present experiments demonstrate that ad libitum drinking water administration of a protein antigen is as effective as i.p. administration or gavage routes of antigen exposure and potentially describe a novel approach to immunization of commercial poultry with purified protein antigens.