Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured in a monolayer. Induction of tyrosine aminotransferase (TAT), which is a hepatocyte-specific function, was enhanced by the non-parenchymal cells, A soluble factor was concluded to be involved in the enhancement because a conditioned medium prepared from non-parenchymal cells was also stimulatory. A hierarchical co-culture, in which hepatocytes and non-parenchymal cells were separated by a collagen layer and which was designed to mimic the in vivo microenvironment of the liver for the long-term maintenance of liver functions was carried out. In this configuration, the level of TAT induction was significantly higher than in the monolayer co-culture. The soluble factor could diffuse through the collagen layer without dispersing into the medium to effectively reach and stimulate hepatocytes. The stimulatory effect was dependent on both the concentration of collagen and the number of non-parenchymal cells. Among the non-parenchymal cells, Kupffer cells were confirmed to be responsible for the enhancement. When cultured alone in a monolayer, hepatocytes completely lost their TAT induction ability within three days, whereas hepatocytes co-cultured with non-parenchymal cells, both in a monolayer and hierarchically, continued to maintain this ability well beyond three days. In particular, a hierarchical co-culture significantly extended the longevity of the hepatocyte function. Effectiveness of the hierarchical co-culture was also shown in the ability of urea synthesis by hepatocytes. The employment of non-parenchymal cells together with hepatocytes in a hierarchical configuration is thus considered to be a promising means of constructing an effective bioartificial liver support.