Dendritic cells (DCs) are key antigen-presenting cells for stimulating immune responses and they are now being investigated in clinical settings. Although defined as lineage-negative (Lin⁻) HLA-DR⁺ cells, significant heterogeneity in these preparations is apparent, particularly in regard to the inclusion or exclusion of CD14⁺, CD16⁺, and CD2⁺ cells. This study used flow cytometry and a panel of monoclonal antibodies (mAbs), including reagents from the 7th Leukocyte Differentiation Antigen Workshop, to define the cellular composition of 2 standardized peripheral blood mononuclear cell (PBMCs)–derived Lin⁻ HLA-DR⁺preparations. Lin⁻ cells were prepared from PBMCs by depletion with CD3, CD14, CD19, CD11b, and either CD16 or CD56 mAbs. Analysis of the CD16-replete preparations divided the Lin⁻ HLA-DR⁺ population into 5 nonoverlapping subsets (mean ± 1 SD): CD123 (mean = 18.3% ± 9.7%), CD1b/c (18.6% ± 7.6%), CD16 (49.6% ± 8.5%), BDCA-3 (2.7% ± 1.4%), and CD34 (5.0% ± 2.4%). The 5 subsets had distinct phenotypes when compared with each other, monocytes, and monocyte-derived DCs (MoDCs). The CD85 family, C-type lectins, costimulatory molecules, and differentiation/activation molecules were also expressed differentially on the 5 Lin⁻HLA-DR⁺ subsets, monocytes, and MoDCs. The poor viability of CD123⁺ DCs in vitro was confirmed, but the CD16⁺ CD11c⁺ DC subset also survived poorly. Finally, the individual subsets used as stimulators in allogeneic mixed leukocyte reactions were ranked by their allostimulatory capacity as CD1b/c > CD16 > BDCA-3 > CD123 > CD34. These data provide an opportunity to standardize the DC populations used for future molecular, functional and possibly even therapeutic studies.