Mechanisms involved in tumor necrosis factor (TNF)-alpha signal transduction are incompletely understood. In some circumstances, TNF may use a signal transduction pathway involving hydrolysis of sphingomyelin to ceramide and stimulation of a ceramide-activated protein kinase. In HL-60 cells, TNF rapidly activates this pathway and induces monocytic differentiation. Here, we demonstrate that treatment of HL-60 cells with TNF selectively increases tyrosine phosphorylation of p42 mitogen-activated protein kinase (p42mapk) and stimulates its enzymatic activity. Induction of p42mapk phosphorylation was time- and dose-dependent and closely paralleled activation of sphingomyelin hydrolysis. Direct engagement of the sphingomyelin signal transduction pathway by addition of bacterial sphingomyelinase led to MAP kinase activation. The time course of p42mapk phosphorylation in the sphingomyelinase-treated cells was similar to that of TNF, with maximal response occurring at 5 min. A maximal concentration of sphingomyelinase (0.01 unit/ml) was more potent than TNF at inducing MAP kinase enzymatic activity (2.6-fold) and phosphorylation of MAP kinase and tyrosine. The cell-permeable ceramide analogs, C2- and C6-ceramide, which mimic effects of TNF, also induced p42mapk phosphorylation within seconds. These studies indicate that the sphingomyelin pathway can regulate MAP kinase activity and suggest that MAP kinase activation by this mechanism may be involved in TNF-induced signal transduction.