Dendritic cells (DC) are the major APC capable of stimulating resting T cells in human peripheral blood (PB). Recent evidence suggested that various subsets of DC and monocytes might circulate in human PB, but their exact phenotype and function had not been delineated. We have previously characterized a population of human PB DC precursors that express the myeloid marker CD33, but not the monocyte marker CD14. To identify and characterize further functional myeloid APC subsets, triple color FACS analysis and sorting was used. A CD33dimCD14dimCD16+ monocyte subset, with similar APC function but less efficient accessory function than CD14bright monocytes, was isolated. In addition to the CD33+CD14dimCD16- DC precursors, a smaller population (0.1 to 0.2% of PBMC) of CD33brightCD14dimCD16- cells with potent APC function was identified. This DC population expressed greater amounts of MHC class II, adhesion, and accessory molecules, and demonstrated a greater costimulatory capacity when freshly isolated than CD33dimCD14dim DC precursors, and therefore had the characteristics of mature, possibly tissue-derived DC. When freshly isolated, however, these DC did not express B7, and up-regulation of accessory function occurred after in vitro differentiation. These data demonstrate multiple circulating myeloid accessory and APC subsets in human PB. Phenotypic and functional differences suggest that they are at different stages of differentiation, and have specialized roles in Ag presentation in vivo. Furthermore, full functional DC differentiation, associated with B7 expression and the capacity to activate T cells maximally, is likely to occur only in specific physiologic circumstances.