The introduction and expression of allogeneic MHC class I genes in tumors can generate tumor-specific immunity which subsequently results in the regression of parental tumors. Immunization of naive (AKR/J x C57BL/6)F1 mice with H-2Kb-transformed K36 tumor cells was found to render recipient mice immune to a subsequent challenge by parental K36 tumor cells. Two types of cytotoxic T effector cells were demonstrated in these immune mice. One of the cytotoxic effector cells generated against the K36 tumor cells is the conventional CD3+ cells, and these account for approximately one-third of the total observed tumor-specific cytotoxicity in vitro. The other cytotoxic effector cell generated following the immunization of (AKR/J x C57BL/6)F1 mice with the H-2Kb-transformed K36 cells had the CD3-/Thy-1+ phenotype, and accounted for the remaining two-thirds of the observed tumor-specific cytotoxicity in vitro. These CD3-/Thy-1+ cells can lyse parental K36 tumor cells in a tumor-specific fashion, and tumor-specific immunity can be adoptively transferred to naive animals via the CD3-/Thy-1+ cells. In contrast to CD3+ CTL, CD3-/Thy-1+ cells express CD45RBlow, Ly-6Chigh, and HSA molecules. Although the CD3-/Thy-1+ cells can be activated in vitro by IL-2, TPA, and ionomycin, they cannot be propagated in vitro. The CD3-/Thy-1+ cells undergo apoptosis following prolonged culture in vitro. At present, the exact mechanism(s) by which CD3-/Thy-1+ cells can mediate tumor-specific cell lysis in the absence of identifiable T cell receptor molecules is unknown; nevertheless, these data suggest the existence of a novel T cell type to combat tumors.