The relative ability of TNF and lymphotoxin (LT) to inhibit the growth of five human tumor cell lines was examined both in the presence and absence of IFN-gamma. Two adenocarcinoma lines, HT-29 and SK-CO-1, were 20- and 320-fold more sensitive to the inhibitory effects of TNF and LT in 3- to 4-day proliferation assays. In contrast, the breast carcinoma line BT-20 showed only a one- to twofold difference. The MCF-7 and ME-180 cell lines exhibited intermediate behavior. These results parallel the reported disparate potencies of TNF and LT in their effects on endothelial cells, hematopoietic development and their abilities to sustain a mixed lymphocyte response. Radiolabeled TNF binding studies showed two classes of receptors (Kd 0.04 to 0.15 nM and 0.2 to 1.0 nM) on the highly sensitive SK-CO-1 line. HT-29 cells also appeared to possess some high affinity-binding sites, whereas the BT-20 line completely lacked the high affinity form. Thus the presence of high affinity-binding sites correlated with increased sensitivity to the antiproliferative effects of TNF. Cold TNF competed with the binding of radiolabeled human TNF three- to fivefold better than LT for binding to all three lines. These relatively small differences between the TNF and LT receptor-binding characteristics are insufficient to explain the dramatic disparity in their antiproliferative properties. Likewise, the absolute concentrations of the unlabeled cytokines necessary to block the binding of 125I-TNF were 10- to 150-fold higher than the levels necessary to elicit the biologic response. Thus, the receptor binding data conflict with the growth inhibitory effects. This discrepancy is discussed in terms of either separate receptors for TNF and LT or more complex phenomena such as receptor cooperativity possibly resulting from multivalent interactions with the trimeric form of TNF.