The functional roles of Cdc2 and checkpoint kinase 1 (Chk1) in synergistic interactions between 7-hydroxystaurosporine (UCN-01) and mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors [e.g., 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide (PD184352)] were examined in human multiple myeloma cells in relation to MEK1/2/ERK1/2 activation and lethality. Time course studies revealed that MEK1/2/extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation preceded Cdc2 dephosphorylation (Tyr15) after UCN-01 exposure. Furthermore, enforced expression of Cdc2 or small inducible RNA (siRNA)-mediated Cdc2 knockdown failed to modify ERK1/2 activation status in either the presence or absence of UCN-01, arguing against a causal relationship between these events. However, ectopic expression of Cdc2 sensitized cells to the lethality of UCN-01/MEK inhibitor regimen, whereas Cdc2 knockdown by siRNA significantly diminished the lethal effects of this combination. Conversely, Chk1 knockdown by siRNA enhanced lethality mediated by UCN-01/PD184352. It is interesting that Chk1 knockdown reduced basal ERK1/2 activation and antagonized the ability of UCN-01 to activate ERK1/2. Finally, ectopic expression of constitutively active MEK1 significantly protected cells from the UCN-01/MEK1/2 inhibitor regimen without modifying Cdc2 activation status. Together, these findings indicate that although UCN-01-mediated Chk1 inhibition and Cdc2 activation are unlikely to be responsible for MEK1/2/ERK1/2 activation, both of these events contribute functionally to enhanced lethality in cells coexposed to MEK inhibitors. They also suggest a role for Chk1 in UCN-01-induced ERK1/2 activation, implying the existence of a heretofore unrecognized link between Chk1 and ERK1/2 signaling.