Normal mouse sera were tested for cytotoxic antibody to surface antigens of cultured monolayer cells infected with AKR-derived ecotropic MuLV, xentropic MuLV, or dualtropic MCF 247 MuLV. Antibody to ecotropic MuLV-infected cells was found in a proportion of C57BL/6, C3Hf/Bi, AKR-Fv-1b, and (C3Hf/Bi X AKR)F1 mice, but not AKR or (AKR X C3Hf/Bi)F1 mice. Antibody to xenotropic MuLV-infected cells was virtually restricted to C57BL/6 mice. Antibody to MCF 247-infected cells was found in all strains tested, including AKR mice. Absorption analysis of (C3Hf/Bi x akr)f1 and AKR-Fv-1b sera with selective reactivity for MCF 247-infected cells showed that these sera recognize distinctive antigens on MCF 247-infected cells that are not present on ecotropic or xenotropic MuLV-infected cells. The transplantable AKR spontaneous leukemia AKSL2 was found to be uniquely sensitive to the cytotoxic action of naturally occurring antibody to MCF 247-related antigens and absorption tests with AKSL2 as the target cell and sera from a single AKR-Fv-1b mouse have permitted the definition of a new MuLV-related cell surface antigen, which has been designated G(AKSL2). Thymocytes from young mice of high leukemia-incidence strains (AKR, C58, and PL) express G(AKSL2), whereas thymocytes from 12 other strains do not. In AKR mice, the antigen is expressed in higher amounts on cells from thymus and bone marrow than on spleen cells. All AKR spontaneous leukemias tested express G(AKSL2), as did three MuLV-induced leukemias arising in G(AKSL2)- strains. Five X-ray-induced leukemias of G(AKSL2)- strains were G(AKSL2)-, as were MuLV+ and MuLV- chemically induced sarcomas. In the limited survey conducted to date, natural antibody to G(AKSL2) has been restricted to strains expressing G(AKSL2) in their normal tissues: AKR, AKR congenic mice AKR-Fv-1b and AKR hybrid mice (C3Hf/Bi x akr)f1 and (C57BL/6 X AKR)F1. In vitro G(AKSL2) induction tests involving MuLV infection of cultured monolayer cells showed that 8 of 12 newly isolated dualtropic MuLV shared the property of G(AKSL2) induction with the prototype MCF MuLV, MCF 247. Of the 12 ecotropic MuLV tested, only the N-tropic MuLV isolated from a leukemia originally induced by Passage A Gross virus induced G(AKSL2). The xenotropic and amphotropic MuLV isolates tested lacked G(AKSL2) inducing activity. Recognition of the g(aksl2) system provides a way to trace the origin and natural history of a class of dualtropic MCF MuLV in the mouse and to determine whether natural antibody to G(AKSL2) plays a role in AKR leukemogenesis.