Lymphocytes from BALB/cCrgl mice react to mouse mammary tumor virus-associated antigen(s) when tested in in vitro blastogenic transformation assays. These mice have a low incidence (less than 1%) of spontaneous mammary tumors and are free from complete mammary tumor virions. We have studied the nature of the lymphoid cells mediating the lymphocyte transformation reaction to purified mammary tumor virus. With the use of nylon wool columns, the responder cells were found to belong to the nylon-adherent population. The T-lymphocytes were not stimulated by mammary tumor virus even in the presence of added macrophages. These results were reconfirmed with treatments of spleen cells with either anti-surface immunoglobulin and complement or anti-Thy 1 antigen and complement. Thus, B-cells seem to be the lymphoid population responsive to mammary tumor virus-associated antigen(s) in the spleen of BALB/cCrgl. The cause of this reactivity may be a result of any of the following: (a) horizontal transmission; (b) activation of spleen cells by viral host cell contaminants in mammary tumor virus preparations; (c) a nonspecific mitogenic reaction exerted by the virus in the system; (d) sensitization to mammary tumor virus-associated antigen(s) due to the expression of an endogenous virus. We present here data arguing against the first three possibilities. In recent work, we found evidence supporting the expression of mammary tumor virus-related antigen(s) on lymphoid cell surfaces of BALB/cCrgl. From these studies, we propose that the responses seen in our in vitro assays may represent a sensitization event resulting from exposure to an endogenous mammary tumor virus gene product.