Preclinical data are reported that support a human immunodeficiency virus (HIV) vaccine strategy using recombinant canarypox-HIV vectors (ALVAC-HIV) to load human dendritic cells (DCs) with HIV antigens. Clinical-grade DCs were infected with good manufacturing practice-grade ALVAC-HIV vaccine constructs. ALVAC infection, HIV gene expression, and DC viability and function were monitored by use of immunohistochemistry, flow cytometry, blastogenesis assays, antigen-specific interferon (IFN)-γ enzyme-linked immunospot assay, and enzyme-linked immunosorbent assay protein detection. The vaccines infected both immature and mature DCs, and intracellular HIV-1 Gag protein was detected within hours. ALVAC-HIV induced DC maturation that was mediated by tumor necrosis factor-α and induced DC apoptosis that was directly related to the length of vaccine exposure. Of importance, the infected DCs remained functional in T cell stimulation assays and induced HIV antigen-specific CD8+ T cell production of IFN-γ from cells of HIV-1-infected individuals. These data support an ongoing HIV vaccine trial comparing conventional vaccine delivery routes with ex vivo vaccine-loaded autologous DCs for immunogenicity in HIV-1-uninfected volunteers.