Background: Toll like receptor (TLR) agonists may enhance vaccination or direct immune activation at the tumor microenvironment. This trial evaluates the biologic and clinical effects of Resiquimod, a TLR 7/8 agonist that can activate both myeloid (mDC, TLR 8) and plasmacytoid (pDC, TLR 7) dendritic cells, in patients with advanced stage melanoma. Methods: Class I HLA-A0201+ subjects with in-transit melanoma metastases or high risk for recurrence were vaccinated weekly with peptide vaccination (class I restricted peptide GP100_209-2m and, if HLA-DP4+, also with class II restricted peptide MAGE-3_243-258). Subjects were randomized 1:1 to receive Resiquimod as an adjuvant applied to the GP100 vaccination site. Subjects with in-transit disease were thereafter treated with resiquimod topically on half of the target lesions. Results: All patients (n = 47) underwent GP100_209-2m vaccination, a majority (39) also received the MAGE-3_243-258 peptide. The type I interferon-inducible genes (Mx A and IRF7), IFNg, and IP-10 RNA expression were up-regulated only in vaccination sites treated with Resiquimod (each p < 0.01) , demonstrating pDC activation (Type I interferon) and possibly T and NK cell activation (IFNg and IP-10). Nineteen subjects had in-transit disease at entry into the trial. In response to peptide vaccination alone, tumor regression was more likely in patients who received Resiquimod at the vaccination site (group A) compared to those who did not (group B). (4/9 vs 0/10, p = 0.033). In group A, 5 patients continued treatment with Resiquimod topically on the tumors, and all had tumor response (4PR, 1CR). In group B, 5 continued to tumoral resiquimod and 3 had regression (3 PR). Conclusions: Resiquimod increases Type I interferon and IFNg at the peptide vaccination site by activation of pDC/mDC and increases the antitumor response sufficiently to mediate regression of in-transit melanoma metastasis. Resiquimod on in-transit melanoma, in vaccinated hosts, drives regression of metastases, regardless of previous exposure at vaccination. Clinical trial information: NCT00960752