Hepatic progenitor cells (HPCs) have been characterized in several drug-treated rodent models and in the fetal liver; however, their properties have not been fully clarified in the normal adult liver, presumably because of their relatively small population and the existence of mature hepatocytes. In an attempt to resolve this issue, we developed a new enrichment system for HPCs using their cell aggregate formation properties. Nonparenchymal cells (NPCs) derived from enzymatically digested liver cells in normal adult mouse liver were treated in a hypoxic 2-hour suspension culture under constant shaking. This procedure resulted in cell aggregate formation and almost complete elimination of mature hepatocytes. Cell aggregates were formed only in Ca2+-containing medium, suggesting cadherin-dependent cell-cell adhesion. In these cell aggregates, 95% consisted of vascular endothelial cells that expressed VE-cadherin. The remaining 5% consisted of rapidly proliferating, small epithelial cells that expressed α-fetoprotein (AFP), E-cadherin, and albumin but not cytokeratin 19 (CK19), α-smooth muscle actin, or VE-cadherin. These results are consistent with an immature hepatic cell phenotype. When these immature hepatic cells were cultured with 10−7 mol/L dexamethasone and 1% dimethyl sulfoxide, the de novo expression of mature hepatocyte markers such as tryptophan-2,3-dioxygenase (TO) was induced concomitantly with the induction of morphologic characteristics such as mitochondria- and peroxisome-rich cytoplasm and bile canaliculi formation. In conclusion, our methodology allows the enrichment of immature hepatic cells from the normal adult mouse. These cells are capable of growth and maturation along the hepatocyte lineage, indicating that these cells are HPCs.