The goal of this study is to determine whether SKI-606, a Src/abl inhibitor, potentiates chk1 inhibitor UCN-01 to induce apoptosis in multiple myeloma cells, and what mechanism maybe involved. We found that the co-exposure of human myeloma cells (e.g., U266, RPMI8226, MM.1S and its dexamethasone-resistant counterparts MM.1R) to minimally toxic concentration of SKI-606 (e.g., 1-2 ƒÝM) and UCN-01 (e.g., 100-150 nM) resulted in dramatic increase in mitochondrial damage and apoptosis. In our previous reports, it has been well demonstrated that activation of Ras/Raf/MEK/ERK pathway represents a critical cytoprotective response in cells exposed to UCN-01. Moreover, Src is required to activate this pathway by growth factors and cytokines. To this end, we examined whether the Src inhibitor enhances UCN-01 lethality through interruption of Ras/Raf/MEK/ERK signaling cascade. Firstly, it was found that co-adminstration of SKI-606 markedly diminished ERK phosphorylation/activation induced by UCN-01, accompanied by an increase in cdc2 activation. Furthermore, myeloma cells with ecpotic expression of either active mutant Ras (Q61L) or constitutive active MEK1 were significantly resistant to combined treatment with SKI-606 and UCN-01, indicating Src inhibition acts upstream of Ras/Raf/MEK to potentiate UCN-01 lethality. Conversely, stable expression of dominant-negative mutant Ras (S17N) markedly sensitized myeloma cells to this combination regimen. Lastly, ectopic expression of kinase inactive (K297R) or dominant-negative (K296R/Y528F) mutant Src blocked ERK activation by UCN-01 and thereby sensitized myeloma cells to UCN-01. Together, these findings indicate that Src inhibitors act through a Ras/Raf/MEK-dependent mechanism to prevent ERK activation in UCN-01-treated cells, resulting in the synergistic induction of cell death.