Little is known regarding the identification, classification, and function of class II MHC+ dendritic cells in the perivasculature of human connective tissues, such as the dermis. We developed a method for preparing papillary dermal cell suspensions from human keratome strips. Among the class II MHC+ populations of the dermis identified using triple color flow cytometry, cells of monocyte/macrophage lineage (CD45+ CD1- CD11b+ CD11clo-mid CD32+ CD36+ or - CD11a-) and mesenchymal cells of non-bone marrow origin (CD45-) were identified and characterized. Another distinct class II MHC+ subset was identified, which expressed a number of features analogous to epidermal Langerhans cells (LC) and other dendritic APC. These were a numerically minor population comprising only 2.7% +/- 1% (n = 7) of dermal cells. Like LC, they express HLA-DR, CD45, CD1a (albeit at a lower level of expression), CD1c, and CD32 and lack constitutive CD11a or ICAM-1. In contrast to LC, this dermal CD1a+CD1c+ subset expresses CD1b, CD11b, a higher level of CD11c, and intracytoplasmic factor XIIIa. Alloantigen presentation by unfractionated dermal cells was reduced by prior removal of this CD1b+ subset to the same degree achieved by removal of the entire DR+ population (20% of dermal cells), indicating that this was the critical DR+ subset. Cocultures of CD4+ T lymphocytes with cells sorted by flow cytometry into CD1c+DR+, CD1c-DR+ and DR- dermal cell subsets positively identified the CD1c+DR+ population as the most potent of potential APC subsets in human dermis. Thus, in distinction to other dermal macrophage and mesenchymal subsets with elongate morphology, the CD1aloCD1b,c+CD11c(hi)CD11b+CD32+DR+ population in human dermis is highly analogous to cells of LC/dendritic APC lineage in its phenotype and in its exclusive ability to potently present Ag to T lymphocytes. These studies identify and characterize the APC subset most potent in inducing activation of T cells initially entering the perivasculature of human dermis to be of LC/dendritic APC, and not tissue macrophage, lineage.