Three distinct dendritic cell (DC) subsets capable of stimulating allogeneic naive T cells were isolated from human thymus. The most abundant subset was represented by plasmacytoid DCs (pDCs), which secreted high amounts of IFN-alpha upon stimulation with inactivated influenza virus and thus likely correspond to the recently identified peripheral blood natural IFN-alpha/beta-producing cells (IPCs). Like those latter cells, thymic pDCs had distinctive phenotypic features (i.e., Lin(-), HLA-DR(int), IL-3R alpha(hi), CD45RA(hi), CD11c(-), CD13(-), and CD33(lo)) and developed into mature DCs upon culture in IL-3 and CD40L. Of the two other DC subsets, one displayed a phenotype of immature myeloid DCs (imDCs) (HLA-DR(int), CD11c(+), CD13(+), CD33(+)), and the other represented HLA-DR(hi) CD11c(+) mature DCs (mDCs). Since they also expressed DC-LAMP, these mDCs appear to correspond to interdigitating dendritic cells (IDCs). Thymic pDCs, but not myeloid imDCs, strongly expressed lymphoid-specific transcripts such as pre-T alpha, lambda-like, and Spi-B, thereby suggesting a possible lymphoid origin. The detection of Spi-B mRNA, not only upon in vitro maturation of pDCs, but also in freshly purified IDCs, suggests that in vivo pDCs may differentiate into IDCs.