Background: The cancer testis antigen NY-ESO-1 (ESO) has been evaluated as a biomarker and a therapeutic immunologic cancer target. Preliminary data from a phase I clinical trial of ESO and ISCOMATRIX adjuvant (ESO vaccine) suggested this treatment could prevent relapse in patients (pts) with high risk resected malignant melanoma (MEL). Methods: We compared ESO vaccine to adjuvant alone in a randomized double-blind phase II study in pts with MEL. Pts with resected stage IIc, IIIb, IIIc, or IV melanoma expressing ESO were randomized 1:1 to ESO vaccine or adjuvant x3 q4w followed by a 4th dose at 6m. Primary endpoint was rate of RFS at 18m in the intent-to-treat (ITT) population and two per protocol (PP) populations, consisting of all relapses regardless of location. Secondary objectives included RFS and Overall Survival (OS) over the entire period of observation (study defined plus off-study follow-up), safety and ESO immunity. Results: Of 111 pts screened with ESO-expressing MEL, 110 comprised the ITT population, with 56 randomized to the ESO vaccine arm and 54 to adjuvant (96% and 87%, respectively, were at stage ≥IIIa at entry). There were no significant differences between the two arms for the ITT population during the 18m observation period for median time to relapse, 139 vs 176 days(p=0.296), or relapse rate, 27 (48.2%) vs 26 (48.1%) (HR=0.913; 95% CI, 0.402 to 2.231), respectively. RFS and OS for the entire period of observation were similar between the two arms. After the third immunization, a significantly larger percentage of pts in the ESO vaccine arm developed a strong positive antibody (Ab) & cellular immune response (IR) to ESO (p<0.001); this difference remained for the duration of the study. A total of 17 out of the 31 pts (55%) who had a vaccine-induced IR (Ab against ESO) and no pre-existing immunity were relapse-free at the end of 18m on study. Conclusions: The ESO vaccine was safe, well tolerated, elicited strong Ab and cellular responses but did not affect relapse free survival. Optimal efficacy may require combining it with other immunotherapy modalities. Clinical trial information: NCT00199901.