Response to treatment with imatinib mesylate has been associated in preclinical models with the inhibition of two signaling pathways that promote cellular survival - the phosphatidylinositol 3-kinase/AKT pathway and the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) pathway. We sought to evaluate the extent of inhibition of these two pathways in metastatic melanoma specimens from patients treated with imatinib. Metastatic melanoma tumor samples were obtained before and during the second week of imatinib treatment from patients enrolled in a phase II study. A tissue microarray was constructed using formalin-fixed, paraffin-embedded tissues, and immunohistochemical analysis was performed using standard techniques to detect phosphorylated (p) ERK1/2 and pAKT expression. Of 21 patients who were treated with imatinib, tumor samples adequate for analysis were available both at baseline and during the second week of treatment from 10 patients for pERK1/2 expression and from nine patients for pAKT expression. No consistent pattern of change in pAKT or pERK expression after treatment with imatinib was observed. No apparent correlation between the clinical benefit of imatinib treatment and changes in pAKT and pERK1/2 expression was observed. A better understanding of the AKT and mitogen-activated protein kinase pathways is needed to optimize the clinical benefit of targeted therapy, such as imatinib.