The maintenance of differentiated hepatocyte phenotype in vitro depends on several factors-in particular, on extracellular matrix interactions, for example, with three-dimensional (3D) matrices. Alginate hydrogel provides the cells with a good extracellular matrix due to the formation of a massive capsule with semi-permeable properties that allows for diffusion of the medium components into the cells as well as efficient waste product elimination. Simultaneously, alginate protects the cells from shear stress caused by the hydrodynamics when cultured in stirred systems such as bioreactors. We have previously developed a hepatocyte aggregate 3D culture system in a bioreactor where improved hepatocyte functionality could be maintained over longer periods (21 days). In this work, ultra-high-viscosity alginate was used for hepatocyte aggregates entrapment. Hepatocyte biotransformation (phase I and II enzymes), CYP450 inducibility, and secretory capacity (albumin and urea production) were monitored. The analyses were performed in both spinner vessels and bioreactors to test the effect of the pO(2) control, unavailable in the spinners. Performance of alginate-encapsulated hepatocyte aggregates in culture was compared with nonencapsulated aggregate cultures in both bioreactor (controlled environment) and spinner vessels. For both culture systems, hepatocytes' metabolic and biotransformation capacities were maintained for up to 1 month, and encapsulated cells in bioreactors showed the best performance. In particular, albumin production rate increased 2- and 1.5-fold in encapsulated aggregates compared with nonencapsulated aggregates in bioreactor and spinner vessels, respectively. Urea production rate increased twofold in encapsulated cultures compared with nonencapsulated cells, in both bioreactor and spinner vessels. Similarly, in both the bioreactor and the spinner system, cell encapsulation resulted in a 1.5- and 2.8-fold improvement of hepatocyte 7-ethoxycoumarin and uridine diphosphate glucuronosyltransferases (UGT) activities, respectively. For all parameters, but for UGT activity, the bioreactor system resulted better than the spinner vessels; for UGT activity no difference was observed between the two. Furthermore, both encapsulated and nonencapsulated 3D culture systems were inducible by 3-methylcholanthrene and dexamethasone. The encapsulated systems consistently showed improved performance over the nonencapsulated cells, indicating that the protection conferred by the alginate matrix plays a relevant role in maintaining the hepatocyte functionalities in vitro.