Abstract The 51Cr release cytotoxicity assay was used to evaluate whether self-reacting clones of T cells develop in vitro when normal spleen cells are cultured with irradiated syngeneic cells. This phenomenon has previously been described as autosensitization and has been thought to be due to reactivity against self-determinants. It was shown, however, in this present study that the autosensitization phenomenon differs qualitatively and quantitatively from specific immune induction by oncofetal antigens and tumor-associated antigens. A variety of tumor cell lines of different H-2 types are lysed by autosensitized cells, and the lysis appears to be nonspecific and not restricted to identity at the major histocompatibility complex (MHC). Induction of autosensitization follows the same kinetic pattern, and lysis is semilogarithmically related to effector cell to target ratio as in other systems. Analysis of the specificity of the phenomenon by competitive inhibition with tumor cells or congenic derived spleen cells did not, however, indicate any reactivity for self-MHC determinants, but rather showed that a wide range of culture tumor cells were all capable of inhibiting lysis. Since it was further shown that the level of autosensitization was markedly dependent on the batch of fetal calf serum, it is provisionally concluded that this phenomenon does not represent specific T cell activation to self-determinants, but rather reactivity against minor antigens in the system, possibly a cell-bound fetal calf serum component.