Hepatocytes were immobilized on a porous gelatin sponge (gelfoam) support. The morphology of the hepatocytes immobilized on the support was close to that in vivo. At some parts of the support, the spheroids of hepatocytes could be observed. To examine the liver-specific functions of hepatocytes on gelfoam, the levels of albumin and bile acids secreted into medium were assessed. In the culture with gelfoam support, the secretion of albumin and bile acids was stable over the course of 12 days, longer than that in collagen-gel culture. To further elucidate the function of hepatocytes immobilized on gelfoam, the metabolic activities of hepatocytes, as measured by the competency of removal of ammonium ions and the synthesis of urea-N were determined. The rates of ammonium removal and urea-N synthesis were comparable with those in conventional monolayer culture. These data demonstrated that gelfoam was a suitable support for the in vitro culture of hepatocytes.