Poxviruses including canarypox (ALVAC) and vaccinia viruses have, in recent years, received considerable attention as live vectors for the development of vaccines against infectious diseases such as AIDS, malaria, and tuberculosis. However, the cellular targets for viral infection within the human immune system and the consequences of infection for cells involved in the generation of immune responses have not been clearly delineated. Using recombinant enhanced green fluorescence protein (EGFP)-expressing ALVAC and vaccinia viruses, we have focused here on a side-by-side comparison of ALVAC and vaccinia virus tropism for cells from human peripheral blood and bone marrow. Both ALVAC and vaccinia viruses showed a strong bias toward monocyte infection. ALVAC minimally infected CD19+ B cells and was unable to infect ex vivo NK cells and T lymphocytes, whereas vaccinia virus could infect B lymphocytes and NK cell populations. Vaccinia virus was also able to infect T lymphocytes at low but detectable levels which could be enhanced upon their activation. Both ALVAC and vaccinia viruses could infect immature monocyte-derived dendritic cells (MDDCs), but only ALVAC infection induced their subsequent maturation. Infection in human bone marrow cells showed that ALVAC infection was restricted to a myelomonocytoid cell-specific CD33+ cell population, while vaccinia virus showed a strong, but not exclusive, preference for these cells.