The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 (MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G 0 , the level of MAPKK activity was observed to affect the kinetics of progression through both the G 1 and G 2 phases of the cell cycle in NIH 3T3 cells. Ectopic expression of dominant-negative forms of MAPKK1, which was previously shown to inhibit G 0 /G 1 progression, was found to also delay progression of cells through G 2 . In addition, treatment of cells with the specific MAPKK inhibitor PD 98059 during a synchronous S phase arrested the cells in the following G 2 phase. These data demonstrate a novel role for the MAPK cascade in progression from G 2 into mitosis in NIH 3T3 cells.