Requirements for the induction of anomalous killer (AK) cells to syngeneic MOPC 104E-KI81 plasmacytoma, and their pattern of reaction were investigated. The AK cells are Thy-1.2 positive and were induced by cocultivation of normal BALB/c spleen cells for 5 days in vitro with mitomycin C-treated MOPC 104E-KI81 cells in RPMI 1640 medium supplemented with fetal calf serum and 2-mercaptoethanol. The pattern of reactivity was investigated by direct cytotoxicity and indirect cold target inhibition assays using a panel of target cells. The results from cold target inhibition show that anomalous killer cells react with various tumor targets in the same preferential order as the results shown in direct cytotoxicity assay, and indicates that the AK cell population may be detecting the same determinants expressed to different degrees on panels of target cells. Kinetic study showed that peak cytotoxic responses were observed on day 5 and day 4, for primary and secondary sensitizing cultures, respectively. Cytotoxicity was not generated when nude mouse spleen cells were used as responder cells, suggesting the essential role of T cells in the induction. Nylon wool-column-purified splenic T cells from mice primed in vivo with intraperitoneal injection of MMC-treated stimulator cells, but not from normal mice, were able to generate AK cells in vitro. These results indicate that generation of AK cells with NK-cell-like reactivity needs inductive factors provided by the immune reaction between sensitized T cells and stimulatory MOPC 104E-KI81 cells. © 1989 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.