Previously we have shown that treatment of ML-1a cells with TNF in the presence of cycloheximide triggers apoptosis within 90 min. In the present report we used this system to investigate the role of ceramide in TNF action. We found that while maximum DNA fragmentation response was induced by 1 nM TNF, the synthetic membrane-permeable C-2, C-6, and C-8 ceramides had no effect even up to 40 microM concentration. To investigate the roles of ceramide in TNF action, we used ((2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxy-phenethyl)amino] propyloxy)benzenesulfonyl))indolizine (SR33557), a potent inhibitor of acidic but not neutral sphingomyelinase (SMase). Even though ceramides by themselves did not mimic TNF, we found that SR33557 inhibited TNF-induced apoptosis and the addition of ceramide reversed this effect, indicating that ceramide generated by acidic SMase is involved in TNF action. The addition of SR33557 to cells at 0 or at 30 min after TNF treatment showed inhibition, but the addition 60 min later had no effect, indicating that an SR33557-sensitive step is located between 30 and 60 min of signal transduction. Since ceramide has been shown to play a role in TNF-mediated activation of nuclear factor-kappa B (NF-kappa B), we examined the effect of SR33557 on this early cellular response of TNF. Surprisingly, this inhibitor of ceramide production was found to have no effect on TNF-mediated NF-kappa B activation, thus suggesting that SR33557-sensitive acidic SMase is not involved in this process. From these results, we concluded that ceramide is needed for certain TNF-mediated cellular responses, but it alone may not be sufficient.