Current biomarkers are unable to adequately predict vaccine-induced immune protection in humans with infectious disease or cancer. However, timely and adequate assessment of antigen-specific immune responses is critical for successful vaccine development. Therefore, we have developed a method for the direct assessment of immune responses in vivo in a clinical setting. Melanoma patients with lymph node (LN) metastases received dendritic cell (DC) vaccine therapy, injected intranodally, followed by [ ¹⁸ F]-labeled 3′-fluoro-3′-deoxy-thymidine ([ ¹⁸ F]FLT) PET at varying time points after vaccination. Control LNs received saline or DCs without antigen. De novo immune responses were readily visualized in treated LNs early after the prime vaccination, and these signals persisted for up to 3 wk. This selective [ ¹⁸ F]FLT uptake was markedly absent in control LNs, although tracer uptake in treated LNs increased profoundly with as little as 4.5 × 10 ⁵ DCs. Immunohistochemical staining confirmed injected DC dispersion to T-cell areas and resultant activation of CD4 ⁺ and CD8 ⁺ T cells. The level of LN tracer uptake significantly correlates to the level of circulating antigen-specific IgG antibodies and antigen-specific proliferation of T cells in peripheral blood. Furthermore, this correlation was not observed with [ ¹⁸ F]-labeled fluoro-2-deoxy-2- d -glucose. Therefore, [ ¹⁸ F]FLT PET offers a sensitive tool to study the kinetics, localization, and involvement of lymphocyte subsets in response to vaccination. This technique allows for early discrimination of responding from nonresponding patients in anti-cancer vaccination and aid physicians in individualized decisionmaking.