The signal transduction pathways activated by tumor necrosis factor α (TNF‐α) 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‐α stimulated a dose‐dependent increase in ERK and p38 MAPK activities that was maximal at 10 min. JNKs were not stimulated by TNF‐α or GM‐CSF. GM‐CSF stimulated ERK activity comparable to that of TNF‐α, 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‐α. GM‐CSF, but not TNF‐α, stimulated wortmannin‐sensitive activation of Raf‐1. TNF‐α 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‐α 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‐α and GM‐CSF to prime the respiratory burst response in human PMNs. J. Leukoc. Biol. 64: 537–545; 1998.