Summary Isoantisera were produced in RIII/Jem mice by repeated administration of C57BL or C3H spleen cells, and their cytotoxic effect on allogeneic Iymphoid cells was analyzed. In the presence of guinea pig complement, both antisera were cytotoxic for spleen cells of all strains of mice studied. Both sera showed a strong cytotoxic effect on thymus cells of all strains of mice tested except those of the AKR/J strain. The RIII anti-C3H serum had only a weak cytotoxic effect on AKR/J thymus cells, while the Kill anti-C57BL serum had no effect at all. This strain distribution indicated that the reaction with thymus cells was primarily caused by antibodies against the θ-C3H antigen present in the RIII antisera studied. Cytotoxicity tests with AKR/J anti-C3H serum and C3H anti-AKR/J serum supported this assumption. The Rill/Jem strain was found to possess the θ-AKR specificity, as did the AKR/J strain, while the AKR/Cum subline possessed the θ-C3H antigen. The capacity of various organs to absorb the cytotoxicity of RIII antisera for thymus cells paralleled their θ-isoantigenicity. On the other hand, the capacity to absorb the cytotoxicity of these antisera for spleen cells reflected mainly their H-2 antigenicity and probably also their Z-isoantigenicity