The signal transduction pathways activated by tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) that lead to priming of polymorphonuclear leukocytes (PMNs) are unknown. The hypotheses that these cytokines stimulate multiple mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and p38 MAPK, and that these MAPKs participate in priming of human PMNs were examined. TNF-alpha stimulated a dose-dependent increase in ERK and p38 MAPK activities that was maximal at 10 min. JNKs were not stimulated by TNF-alpha or GM-CSF. GM-CSF stimulated ERK activity comparable to that of TNF-alpha, but GM-CSF was a less potent stimulus of p38 MAPK activity. The tyrosine kinase inhibitor, genistein, inhibited ERK and p38 MAPK stimulation by both cytokines. The phosphatidylinositol 3-kinase inhibitor, wortmannin, attenuated stimulation of ERKs and p38 MAPK by GM-CSF, but not TNF-alpha. GM-CSF, but not TNF-alpha, stimulated wortmannin-sensitive activation of Raf-1. TNF-alpha and GM-CSF priming of superoxide release stimulated by N-formyl-methionyl-leucyl-phenylalanine was significantly attenuated by the MEK inhibitor, PD098059, and the p38 MAPK inhibitor, SB203580. Incubation with both MAPK inhibitors produced an additive effect. Our data suggest that TNF-alpha and GM-CSF activate ERKs and p38 MAPK by different signal transduction pathways. Both ERK and p38 MAPK cascades contribute to the ability of TNF-alpha and GM-CSF to prime the respiratory burst response in human PMNs.