Plasmacytoid dendritic cells (PDC) are potent producers of alpha interferon (IFN-alpha) in response to enveloped viruses and provide a critical link between the innate and adaptive immune responses. Although the loss of peripheral blood PDC function and numbers has been linked to human immunodeficiency virus (HIV) progression in humans, a suitable animal model is needed to study the effects of immunodeficiency virus infection on PDC function. The rhesus macaque SIV model closely mimics human HIV infection, and recent studies have identified macaque PDC, potentially making the macaque a good model to study PDC regulation. In this study, we demonstrate that peripheral blood PDC from healthy macaques are both phenotypically and functionally similar to human PDC and that reagents used for human studies can be used to study macaque PDC. Both human and macaque PBMC expressed IFN-alpha in response to herpes simplex virus (HSV), the prototypical activator of PDC, as measured by using an IFN bioassay and IFN-alpha-specific enzyme-linked immunospot assays. Similar to human PDC, macaque PDC were identified by using flow cytometry as CD123+ HLA-DR+ lineage- cells. In addition, like human PDC, macaque PDC expressed intracellular IFN-alpha, tumor necrosis factor alpha, macrophage inflammatory protein 1beta/CCL4, and IFN-inducible protein 10/CXCL10 upon stimulation with HSV, all as determined by intracellular flow cytometry. We found that IFN regulatory factor 7, which is required for the expression of IFN-alpha genes, was, similar to human PDC, expressed at high levels in macaque PDC compared to monocytes and CD8+ T cells. These findings establish the phenotypic and functional similarity of human and macaque PDC and confirm the utility of tools developed for studying human PDC in this animal model.