Dendritic cells (DC) in the colon may regulate intestinal immunity but remain poorly characterized. In this study a CD11c+ HLA-DR+ lin−(CD3− CD14− CD16− CD19− CD34−) population has been identified by flow cytometry in cells obtained by rapid collagenase digestion of human colonic and rectal biopsies. These day 0 (d0) CD11c+ HLA-DR+ lin− cells comprised∼ 0.6% of the mononuclear cells obtained from the lamina propria, were endocytically active, and had the phenotype of immature DC; they were CD40+ and expressed low levels of CD83 and CD86, but little or no CD80 or CD25. Similar d0 DC populations were isolated from the colonic mucosa of healthy controls and from both inflamed and noninflamed tissue from patients with Crohn’s disease. The lamina propria also contained a population of cells capable of migrating out of biopsies during an overnight culture and differentiating into mature DC with lower levels of endocytic activity and high cell surface expression of CD40, CD80, CD86, CD83, and CD25. This mature DC population was a potent stimulator of an allogeneic mixed leukocyte (MLR). Overnight culture of cells isolated by enzymatic digestion on d0 yielded DC with a phenotype intermediate between that of the d0 cells and that of the cells migrating out overnight. Overnight culture of colonic cells in which DC and HLA-DR+ lin+ cells were differentially labeled with FITC-dextran suggested that some of the maturing DC might differentiate from HLA-DR+ lin+ progenitors. This study presents the first analysis of the phenotype, maturational status, and migratory activity of human gut DC.