The majority of melanomas show constitutive activation of the RAS-RAF-MAP/ERK kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway. AZD6244 is a selective MEK1/2 inhibitor that markedly reduces tumor P-MAPK levels, but it produces few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the phosphoinositide 3-kinase (PI3K)-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines showed AZD6244-induced activation of AKT. In contrast, sensitive cell lines showed AZD6244 treatment–induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or insulin-like growth factor I receptor blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells and the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients. Cancer Res; 70(21); 8736–47. ©2010 AACR.