Purpose: A decrease in the frequency and activation state of dendritic cells in the sentinel lymph node (SLN) has been observed in early stages of melanoma development. This may hinder the generation of effective antitumor T-cell responses and increase the likelihood of metastatic spread. Immunopotentiation of the melanoma SLN may therefore be a valuable adjuvant treatment option. One way to achieve this is through the use of bacterially derived unmethylated cytosine-phosphate-guanine (CpG) DNA sequences that bind Toll-like receptor 9 and activate plasmacytoid dendritic cells (PDC). CpG-activated PDC, in turn, release IFNα and may thus boost T-cell and natural killer cell responses as well as activate conventional myeloid dendritic cells (MDC). Experimental Design: We studied the effects of preoperative local administration of the CpG B-type oligodeoxynucleotide (ODN) PF-3512676 (formerly known as CPG 7909) on dendritic cell and T-cell subsets in the SLN of 23 stage I to III melanoma patients, randomized to receive intradermal injections of either PF-3512676 or saline (NaCl 0.9%). Results: PF-3512676 administration resulted in bulkier SLN, higher yields of isolated SLN leukocytes, and activation of BDCA-2+CD123+ PDC as well as of CD1a + MDC. In addition, PF-3512676 administration was associated with the presence of a newly identified CD11chi CD123+CD83 +TRAIL+ mature SLN-MDC subset, an increased release of a variety of inflammatory cytokines, and lower frequencies of CD4 +CD25hiCTLA-4+FoxP3+ regulatory T cells in the SLN. Conclusions: These findings point to the possible utility of the conditioning of SLN by PF-3512676 as an adjuvant immunotherapeutic modality for early-stage melanoma.