In this study, a co-culture system of hepatocytes and bone marrow cells (BMCs) was developed and characterized for the expression and maintenance of ammonia metabolism and albumin secretion activities. A culture medium supplemented with epidermal growth factor, insulin, L-proline, hydrocortisone and 20 % (v/v) heat-inactivated fetal bovine serum was developed. In addition to adhesive bone marrow cells, the co-existence of non-adhesive bone marrow cells was effective in expressing liver-specific functions for at least 3 weeks. On the other hand, experiments with Transwell in which cultured cells were separated by a semi-permeable membrane, suggested that soluble factors secreted by BMCs are the key components in the functional enhancement of cells. Furthermore, direct contact between hepatocytes and BMCs enhanced the formation of spheroids and the expression of liver specific functions. These results indicate that this co-culture system is promising in, for example, bioartificial liver, regenerative medicine, and liver function simulator applications. © Wichtig Editore, 2004.