White Rock embryos surgically "bursectomized" at 52--64 h of incubation, and shambursectomized embryos were injected with 10(6) guinea-pig red blood cells on day 12 of incubation, and tested for plaque-forming cells and serum haemagglutinins 3, 5, 7, 10, 15 and 19 days after immunization, i.e. as 15- to 19-day-old embryos and 1- to 10-day-old chickens. The number of natural plaque-forming cells detected by direct or indirect techniques was small in nonimmunized shambursectomized and bursectomized embryos, but increased in very young chickens. The injection of guinea-pig red blood cells induced a significant increase in the number of direct and indirect plaque-forming cells in the spleen of bursectomized and sham-bursectomized embryos and chickens. Agglutination of papain-treated guinea-pig red blood cells and indirect anti-chicken globulin (Coombs) test revealed the presence of natural agglutinins for guinea-pig and for sheep erythrocytes in non-immunized sham-and bursectomized embryos. A small number of sera from nonimmunized bursectomized and sham-bursectomized embryos contained IgM. The immunization with guinea-pig red blood cells increased the antibody production in both bursectomized and sham-bursectomized embryos and chickens. Sham-bursectomized embryos responded better to antigenic stimulation than bursectomized embryos. The injection of guinea-pig red blood produced an enlargement of the spleen only in bursectomized embryos and chickens. The first plasma cells appeared in nonimmunized sham-and bursectomized 6-day-old chickens. The number of plasma cells increased in chickens immunized as embryos. Cytomorphological analysis of the thymus, bone marrow and liver did not reveal apparent differences between bursectomized, sham-bursectomized embryos and very young chickens. It has been postulated that the chicken embryo has an antibody-producing system composed of the bursal and the nonbursal (or accessory "bursal") microenvironment, the latter being bursa-independent. The final microenvironmental network for the formation of Bu lymphocytes is the result of coordinated activities of a variety of intrinsic cellular and humoral factors.