This paper describes an in vitro system for studying the factors that control and regulate the cellular events in the immune response. The authors report that dissociated cell suspensions from normal mouse spleens can be immunized in vitro to heterologous erythrocytes, with the response being primary in nature. The response is measured by the increase in the number of hemolytic plaque-forming cells and by the assay of antibody in the culture supernatants. Critical conditions for culture and immunization include low oxygen tension, gentle agitation, the inclusion of fetal bovine serum, adequate spleen cell density, and daily feeding with a nutritional mixture. The in vitro response closely parallels the in vivo response in terms of size, early kinetics, effect of antigen dose, and the inhibitory effect of passive antibody. However, the in vitro response shows greater discrimination between different homologous erythrocyte antigens and does not appear to be limited by mechanisms that regulate the in vivo response. The authors discuss the implications of these differences and the potential underlying regulatory mechanisms. The system is useful for studying the mechanisms controlling the immune response and has been applied to the study of cell proliferation.This paper describes an in vitro system for studying the factors that control and regulate the cellular events in the immune response. The authors report that dissociated cell suspensions from normal mouse spleens can be immunized in vitro to heterologous erythrocytes, with the response being primary in nature. The response is measured by the increase in the number of hemolytic plaque-forming cells and by the assay of antibody in the culture supernatants. Critical conditions for culture and immunization include low oxygen tension, gentle agitation, the inclusion of fetal bovine serum, adequate spleen cell density, and daily feeding with a nutritional mixture. The in vitro response closely parallels the in vivo response in terms of size, early kinetics, effect of antigen dose, and the inhibitory effect of passive antibody. However, the in vitro response shows greater discrimination between different homologous erythrocyte antigens and does not appear to be limited by mechanisms that regulate the in vivo response. The authors discuss the implications of these differences and the potential underlying regulatory mechanisms. The system is useful for studying the mechanisms controlling the immune response and has been applied to the study of cell proliferation.