Tumor necrosis factor (TNF) is a 17-kDa protein produced by monocytes and a wide variety of other cell types in response to endotoxin and other cytokines. In contrast, lymphotoxin (LT) is a 25-kDa glycoprotein produced only by lymphocytes activated by mitogens. These two cytokines are 28% identical in their amino acid sequences. As they have common cell surface receptors, it is generally assumed that all cellular responses mediated through TNF are also mediated by LT and vice versa. In this report we tested this assumption, comparing the effect of TNF and LT on mediation of early (activation of the transcription factor NF-kappa B) and late (reduction of nitro blue tetrazolium, NBT) cellular responses in the human myelomonoblastic leukemic cell line ML-1a. Both qualitative and quantitative differences were found. LT was found to display 5-10 times more potent antiproliferative effects against murine fibroblasts than TNF. However, in ML-1a cells at concentrations wherein TNF activated NF-kappa B, LT did not. Higher concentrations (1,000-10,000 fold) of LT could activate NF-kappa B, but the activated complex was short lived (less than 1 h versus greater than 6 h when activated by TNF) and required longer treatment (15 min versus less than 5 min). TNF induced NBT-reducing activity in a dose-dependent manner, whereas LT was essentially inactive. Since both TNF and LT have been shown to bind to a common receptor, we tested whether the TNF-induced effects could be blocked by LT. LT inhibited both the early and late TNF-mediated cellular responses. By using receptor-blocking antibodies we found that both p60 and p80 forms of TNF receptors were functional for NBT-reducing activity, but TNF-dependent NF-kappa B activation required only the p60 receptor. Furthermore, we found that both TNF and LT bound with higher affinity to the p80 than to the p60 receptor. Thus, our overall results indicate that there are qualitative and quantitative differences in the action of TNF and LT, and these could be noted quite early in their signaling.