Background: Case control studies have demonstrated that administration of CancerVax, a polyvalent melanoma cell vaccine (PMCV), after complete resection of melanoma metastases produces a significant improvement in disease-free survival (DFS). Because PMCV has no direct cytotoxic effect on melanoma cells, the authors hypothesized that it prolongs survival by enhancing antibody-mediated antimelanoma cytotoxicity. Methods: One hundred melanoma patients participating in a trial of PMCV adjuvant therapy following complete resection of regional node metastases were randomly selected for study. Serum samples obtained immediately before (T0) and 4, 8, 12, and 16 weeks after initiation of PMCV adjuvant therapy were adsorbed with L-14 lymphoblastoid cells and then tested for in vitro complement-dependent cytotoxicity (CDC) against M-14 cells, a melanoma cell line not used in PMCV. CDC was expressed as percentage of total cells (n=10,000) killed. Survival curves were estimated by the Kaplan-Meier method. Statistical analysis was performed by the signed rank sum test, Spearman test, log-rank test, and Cox proportional hazard regression. Results: Median CDC at T0 was 4.5% (range, 0% to 40%). Within 16 weeks after initiation of PMCV therapy, CDC had increased in 82 (82%) patients. The median increase of 7.5% (range, −9% to 39%) represented a highly significant change (signed rank sum test;P=.0001). At a median follow-up of 29 months (range, 6 to 92 months), the maximum increase in CDC (ΔCDC) as a continuous variable was significantly correlated with DFS (P=.0001). Median survival and 5-year DFS were more than 54 months and less than 54%, respectively, for patients with ΔCDC≥10% (n=44) but only 7 months and 14%, respectively, for those with ΔCDC<10% (n=56;P=.0001). Multivariate analysis confirmed ΔCDC as the most significant independent variable associated with DFS following initiation of PMCV therapy (P=.0001). Conclusion: PMCV therapy greatly enhances serum CDC against melanoma cells. This enhancement is directly correlated with DFS following initiation of vaccine therapy.