Purpose: Autophagy consists of lysosome-dependent degradation of cytoplasmic contents sequestered by autophagic vesicles (AV). The role of autophagy in determining tumor aggressiveness and response to therapy in melanoma was investigated in this study.Experimental Design: Autophagy was measured in tumor biopsies obtained from metastatic melanoma patients enrolled on a phase II trial of temozolomide and sorafenib and correlated to clinical outcome. These results were compared with autophagy measurements in aggressive and indolent melanoma cells grown in two-and three-dimensional (3D) culture and as xenograft tumors. The effects of autophagy inhibition with either hydroxychloroquine or inducible shRNA (short hairpin RNA) against the autophagy gene ATG5 were assessed in three-dimensional spheroids.Results: Patients whose tumors had a high autophagic index were less likely to respond to treatment and had a shorter survival compared with those with a low autophagic index. Differences in autophagy were less evident in aggressive and indolent melanoma cells grown in monolayer culture. In contrast, autophagy was increased in aggressive compared with indolent melanoma xenograft tumors. This difference was recapitulated when aggressive and indolent melanoma cells were grown as spheroids. Autophagy inhibition with either hydroxychloroquine or inducible shRNA against ATG5 resulted in cell death in aggressive melanoma spheroids, and significantly augmented temozolomide-induced cell death.Conclusions: Autophagy is a potential prognostic factor and therapeutic target in melanoma. Three dimensional culture mimics the tumor microenvironment better than monolayer culture and is an appropriate model for studying therapeutic combinations involving autophagy modulators. Autophagy inhibition should be tested clinically in patients with melanoma. Clin Cancer Res; 17(10); 3478-89. Ó2011 AACR.