The oval cell is regarded as a compensatory cell in liver injury, and is thought to be equivalent to liver stem/progenitor cells. Oval cells were induced by the 2-AAF/CCl4 dietary method in Fischer 344 rats, and were isolated from excised liver by the collagenase perfusion, enzyme treatment, and cell cloning method. Transmission electron microscopy observation and double immunofluorescence methods were used to characterize the cells. We have developed an in vitro system consisting of three-dimensional collagen and hormonal and cytokine factors. Over 3 weeks, albumin secretion and urea detoxification rate were estimated to assess the biological function of the oval cells cultured in a scaffold. Oval cells cultured in the scaffold demonstrated higher biological functions than did those in a two-dimensional tissue culture plate. The pore structure and collagen in a scaffold may play an important role in fostering the biochemical functions of oval cells. The three-dimensional culture of oval cells could be considered in designing a cell-delivering tool for hepatic disease.