Effector cell types and effector mechanisms of human spontaneous lymphocyte-mediated cytotoxicity (SLMC) were studied in a 44-h microcytotoxicity titration assay. Peripheral blood lymphocytes from cancer patients and controls were used as effector cells either unfractionated or after fractionation by rosetting techniques or affinity chromatography. The possible immunoglobulin dependency of the reactions was studied by incorporation of specific Fab fragments of rabbit anti-human IgG antibodies in the incubation mixtures. Twelve different target cell lines of either high or low sensitivity to SLMC and with or without easily detectable HLA antigens were used. Most of the target cells were cell lines derived from transitional cell carcinoma of the urinary bladder (TCC). Both allogeneic and autologous lymphocyte target cell combinations were tested. Although high- and low-sensitivity target cells differed significantly in susceptibility to lysis, the predominating SLMC was displayed by Fc-receptor-positive lymphocytes in both allogeneic and autologous combinations. Addition of the Fab anti-immunoglobulin reagent to the incubation mixtures resulted in strong inhibition of cytotoxicity regardless of the type of target cells used and in allogeneic as well as in autologous lymphocyte target cell mixtures. However, in some combinations no inhibition was seen and inhibition was usually not complete, suggesting that both immunoglobulin-dependent (i.e., ADCC-like) and immunoglobulin-independent mechanisms were involved in the cytotoxicity reactions. The results of the microcytotoxicity assay were compared with those obtained with aliquots of the same lymphocytes and target cells in an 18-h 51Cr-release assay. While similar results were obtained with high-sensitivity target cells, with low-sensitivity targets and in some autologous combinations the two assay systems registered lymphocyte/target cell interactions which differed with regard to specificity, effector cell type, and immunoglobulin dependency.