BACKGROUND Forty‐eight patients with resected Stages IIA and IIB melanoma were immunized with two tumor antigen epitope peptides derived from gp100209–217 (210M) (IMDQVPSFV) and tyrosinase368–376 (370D) (YMDGTMSQV) emulsified with incomplete Freund's adjuvant (IFA). Patients were assigned randomly to receive either peptides/IFA alone or with 250 μm of granulocyte‐macrophage–colony‐stimulating factor (GM‐CSF) subcutaneously daily for 5 days to evaluate the toxicities and immune responses in either arm. Time to recurrence and survival were secondary end points. METHODS Immunizations were administered every 2weeks × 4, then every 4 weeks × 3, and once 8 weeks later. A leukapheresis to obtain peripheral blood mononuclear cells for immune analyses and skin testing with peptides and recall reagents was performed before and after eight vaccinations. RESULTS Local pain and granuloma formation, fever, and lethargy of Grade 1 or 2 were observed. Transient vaccine‐related Grade III and no Grade IV toxicity was observed. Seventeen of the 40 patients for whom posttreatment skin tests were performed developed a positive skin test response to the gp100 peptide, but only 1 of the 40 patients developed a positive skin test response to tyrosinase. Immune responses were measured by release of interferon‐gamma (IFN‐γ) in an enzyme‐linked immunosorbent assay (ELISA) by effector cells in the presence of peptide‐pulsed antigen‐presenting cells, by cytokine release of IFN‐γ, GM‐CSF, and tumor necrosis factor‐alpha in a Luminex assay, or by an antigen‐specific tetramer flow cytometry assay. Thirty‐four of the 39 patients for whom the ELISA data were performed demonstrated an immune response after vaccination, as did 37 of 42 patients by tetramer assay. Enzyme‐linked immunosorbent assay, Luminex, and tetramer responses in the GM‐CSF/peptide/IFA group were higher than in the peptide/IFA group. Epitope spreading to the MART‐1/MelanA 27‐35 and 26‐35 (27L) epitopes was detected by tetramer assay in 10 patients. Seven of 48 patients experienced disease recurrence with a median of 24 months of follow‐up and 2 patients in this intermediate to high risk group have died. CONCLUSION These data suggest a significant number of patients with resected melanoma mount an antigen‐specific immune response against a peptide vaccine. There is a trend for GM‐CSF to modestly increase the immune response and support further development of GM‐CSF as a vaccine adjuvant. Cancer 2003;97:186–200. © 2003 American Cancer Society. DOI 10.1002/cncr.11045