Dendritic cells (DC) represent potent APCs that are capable of generating tumor-specific immunity. We performed a pilot clinical trial using Ag-pulsed DC as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly injections of DC enriched and activated from their PBMC. DC were cocultured ex vivo with recombinant mouse prostatic acid phosphatase as the target neoantigen. Following enrichment, DC developed an activated phenotype with up-regulation of CD80, CD86, and CD83 expression. During culture, the DC maintained their levels of various adhesion molecules, including CD44, LFA-1, cutaneous lymphocyte-associated Ag, and CD49d, up-regulated CCR7, but lost CD62 ligand and CCR5. In the absence of CD62 ligand, such cells would not be expected to prime T cells efficiently if administered iv due to their inability to access lymphoid tissue via high endothelial venules. To assess this possibility, three patient cohorts were immunized with Ag-pulsed DC by iv, intradermal (id), or intralymphatic (il) injection. All patients developed Ag-specific T cell immune responses following immunization, regardless of route. Induction of IFN-γ production, however, was seen only with id and il routes of administration, and no IL-4 responses were seen regardless of route, consistent with the induction of Th1-type immunity. Five of nine patients who were immunized by the iv route developed Ag-specific Abs compared with one of six for id and two of six for il routes. These results suggest that while activated DC can prime T cell immunity regardless of route, the quality of this response and induction of Ag-specific Abs may be affected by the route of administration.