The role of spleen in local (per anum) and systemic (intravenous = i.v.) immunization was studied by splenectomizing chickens and immunizing them twice with sheep red blood cells (SRBC) and Brucella abortus. The number of germinal centers in spleen and the splenic weight were also recorded. Splenectomy had no effect on the serum titres evoked by the per anum immunization but it affected the anti-SRBC and, to a lesser extent, anti-Brucella titres in serum after i.v. immunization. The effect of splenectomy was less obvious both in the secondary response and when the chickens were younger at operation and the time between splenectomy and immunization was increased. The i.v. immunizations induced germinal centre formation in the spleen whereas immunization per anum did not significantly do so. Also the splenic weight in the i.v. immunized chickens was higher than that in the per anum immunized birds. The present study shows that the spleen is the major site of antibody production against i.v. administered antigens in chickens. Antibody production to antigens applied per anum occurs mainly in other lymphatic organs, most probably in the gut-associated lymphoid tissues.