Primary cultures of human hepatocytes are a reference cellular model, because they maintain key features of liver cells in vivo, such as expression of drug-metabolizing enzymes, response to enzyme inducers, and generation of hepatic metabolites. However, there is a restricted availability of primary hepatocytes, and they show phenotypic instability in culture. Thus, different alternatives have been developed to overcome the culture limitations and to mimic in vivo tissue material. Herein, culture conditions, such as medium composition, impeller type, and cell inoculum concentration, were optimized in stirred culture vessels and applied to a three-dimensional (3D) bioreactor system. Cultures of rat hepatocytes as 3D structures on bioreactor, better resembling in vivo cellular organization, were compared to traditional monolayer cultures. Liver-specific functions, such as albumin and urea secretion, phase I and phase II enzyme activities, and the capacity to metabolize diphenhydramine and troglitazone, were measured over time. Hepatocyte functions were preserved for longer time in the 3D bioreactor than in the monolayer system. Moreover, rat hepatocytes grown in 3D system maintained the ability to metabolize such compounds, as well as in vivo. Our results indicate that hepatocytes cultured as 3D structures are a qualified model system to study hepatocyte drug metabolism over a long period of time. Moreover, these cultures can be used as feeding systems to obtain cells for other tests in a short time.