Previous studies have identified novel lymphoid phenotypes in the adult human liver and provided evidence to suggest that lymphoid differentiation can occur locally in this organ. The aim of this study was to examine the adult human liver for the presence of hematopoietic stem cells that may provide the necessary precursor population for local hematopoietic and lymphoid differentiation. Hepatic mononuclear cells (HMNC) were extracted from normal adult liver biopsy specimens using a combination of mechanical disruption and enzymatic digestion. The stem cell marker CD34 was found on 0.81% to 2.35% of isolated HMNCs by flow cytometry. CD34 ุ HMNCs were positively selected using magnetically labeled beads, and the enriched population was further examined for surface markers characteristically expressed by immature hematopoietic cells and early progenitors. CD45 was expressed by 49% (ุ23%) of CD34 ุ HMNCs, indicating their hematopoietic origin. CD38, one of the first markers to be expressed by developing progenitor cells was found on 50% (ุ22%) of CD34 ุ HMNCs indicating the presence of both pluripotent stem cells and committed precursors. The majority (90%) of CD34 ุ HMNCs coexpressed the activation marker human leukocyte antigen DR, consistent with actively cycling cells. Functional maturation of these hepatic progenitors was shown by the detection of multilineage hematopoietic colony formation after tissue culture. Erythroid (BFU-E), granuloctye-monocyte (CFU-GM), and mixed colonies (CFU-GEMM) were detected after culture of unseparated HMNCs and the enriched CD34 ุ HMNC population; 14.3 ุ 13.2 (mean ุ SD) BFU-E, 3.1 ุ 3.1 CFU-GM, and 0.4 ุ 0.9 CFU-GEMM per 1 ุ 10 5 unseparated HMNCs and 16.0 ุ 9.5 BFU-E and 1.7 ุ 0.9 CFU-GM were identified per 2.4 ุ 10 3 CD34 ุ HMNCs plated. The detection of surface markers characteristic of immature hematopoietic cells and colony formation in tissue culture provides evidence for the presence of hematopoietic stem cells and early progenitor cells in the adult human liver. This would suggest that the adult human liver continues to contribute to hematopoiesis and may be an important site for the differentiation of lymphohematopoietic cells involved in disease states, such as autoimmune hepatitis and graft rejection after liver transplantation. (HEPATOLOGY 1999;29:1193-1198.