Plasmacytoid dendritic cells (pDC) are well-known for their ability to produce large quantities of interferon-alpha (IFN-alpha) in response to viruses. In addition, pDC produce IFN-alpha in response to HSV-infected cells. We demonstrate that both tonsil and PBMC contain pDC that respond to stimulation with HSV either in suspension or in tonsil tissue-fragment culture. We hypothesized that other DC subsets acquire virus in the periphery and deliver the interferongenic signals to the pDC in the draining lymphoid tissue. As a model for pDC/myeloid DC interaction, we studied the interaction of pDC derived from blood with HSV-infected and uninfected monocyte derived dendritic cells (MDDC). Infected, but not uninfected, MDDC induced IFN-alpha in pDC. To further study pDC/infected MDDC interactions, we labeled MDDC with fluorescent cell trackers PKH67 or CFSE prior to infection with HSV and co-cultured with pDC. Cells were then analyzed using conventional and imaging flow cytometry. In addition, we infected MDDC with a GFP-expressing HSV prior to co-culture with pDC. Using traditional flow cytometry, we observed that pDC became fluorescent after co-incubation with uninfected or infected, fluorescently labeled MDDC, indicating that MDDC transferred fluorescent protein and membrane to pDC. By imaging flow cytometry, we observed formation of conjugates between pDC and MDDC as well as transfer and internalization of cellular components from the labeled MDDC by pDC, with preferential uptake from, and association with, infected vs. uninfected MDDC. These studies demonstrate that MDDC infected with HSV are able to stimulate IFN-alpha and chemokine production by pDC through the transfer of cellular materials from the HSV-infected MDDC to the pDC. Together, these observations indicate that heterogeneous populations of DC interact to generate an effective IFN-alpha response.