Granulocyte–macrophage colony–stimulating factor (GM-CSF) enhances immune responses by inducing proliferation, maturation, and migration of dendritic cells (DCs) as well as expansion and differentiation of B and T lymphocytes. The potency of DNA vaccines can be enhanced by the addition of DNA encoding cytokines, acting as molecular adjuvants. We conducted a phase I/II trial of human GM-CSF DNA in conjunction with a multipeptide vaccine (gp100 and tyrosinase) in stage III/IV melanoma patients. Nineteen human leukocyte antigen (HLA)-A*0201(+) patients were treated. Three dose levels were studied: 100, 400, and 800 µg DNA/injection, administered subcutaneously every month with 500 µg of each peptide. In the dose-ranging study, three patients were treated at each dose level. The remaining patients were then treated at the highest dose. Most toxicities were grade 1 injection-site reactions. Eight patients (42%) developed CD8⁺ T-cell responses, defined by a ≥3 SD increase in baseline reactivity to tyrosinase or gp100 peptide in tetramer or intracellular cytokine staining (ICS) assays. There was no relationship between dose and T-cell response. Responding T cells had an effector memory cell phenotype. Polyfunctional T cells were also demonstrated. At a median of 31 months follow-up, median survival has not been reached. Human GM-CSF DNA was found to be a safe adjuvant.