Bioartificial liver devices replace the function of the failing liver, using primary hepatocytes cultured in a bioreactor module. Most devices have been based on cartridge designs, but alternative designs using monolayers of cells in a flat plate bioreactor may be more efficacious. Collagen coating improves the maintenance of hepatocytes on polymeric membranes, and in this article the effect of contact with glycosaminoglycans (GAGs) on the function of hepatocytes was assessed. The effect of two different GAGs, chondroitin-6-sulfate and heparin, in the presence and absence of a cross-linking agent (1,6-diaminohexane [DAH]), on the activities of two major metabolic pathways in hepatocytes (cytochrome P-450-dependent monooxygenase activity, assessed by the hydroxylation of testosterone, and UDP-glucuronosyltransferase activity, assessed by the glucuronidation of kaempferol) cultured on collagen gels and films is presented. Testosterone metabolism was more extensive in cells cultured on collagen films than in cells cultured on gels. The addition of heparin and DAH to collagen gels supported the formation of 6beta-hydroxy, 16alpha-hydroxy, and 2alpha-hydroxy testosterone by cells cultured for 48 h. The extent of glucuronidation of kaempferol was not different when comparing cells cultured on films or gels at the various times in culture; however, the ratio of formation of the two glucuronides formed, M1 and M2, was different. The combination of chondroitin- 6-sulfate and DAH increased glucuronidation of cells cultured for 7 days on both collagen films and gels. This approach may increase the expression of hepatocyte-specific functions in monolayers cultured on membranes in flat plate bioreactors.