This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C 2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C 2-ceramide activated c-Jun NH 2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C 2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21 CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21 CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C 2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C 2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C 2-ceramide-and PMA-stimulated U937 cells to that of cells treated with C 2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C 2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C 2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.