Primary rat hepatocytes were co-cultured with bone marrow cells (BMCs) in a tissue culture-treated culture well (TCW) or a collagen-coated culture well (CCW). Although a medium containing serum was used, the co-cultured cells formed spheroids on the TCW within 2 days, and ammonia metabolism and albumin secretion activities were well maintained for 3 weeks. The co-cultured cells formed a monolayer on the CCW within 2 days, and liver functions were maintained for 3 weeks. The ammonia metabolism activities and albumin secretion activities of co-cultured cells on the CCWs were slightly inferior to those on the TCWs. This co-culture system of hepatocytes and BMCs is an appropriate culture system for the expression and maintenance of liver-specific functions in vitro, and is expected to be applicable to bioartificial liver systems, regenerative medicine, and liver function simulators. © The Japanese Society for Artificial Organs 2005.