We have developed a perfusion bioreactor system that allows the formation of steady state oxygen gradients in cell culture. In this study, gradients were formed in cultures of rat hepatocytes to study the role of oxygen in modulating cellular functions. A model of oxygen transport in our flat-plate reactor was developed to estimate oxygen distribution at the cell surface. Experimental measurements of outlet oxygen concentration from various flow conditions were used to validate model predictions. We showed that cell viability was maintained over a 24-h period when operating with a physiologic oxygen gradient at the cell surface from 76 to 5 mmHg O-2 at the outlet. Oxygen gradients have been implicated in the maintenance of regional compartmentalized metabolic and detoxification functions in the liver, termed zonation. In this system, physiologic oxygen gradients in reactor cultures contributed to a heterogeneous distribution of phosphoenolpyruvate carboxykinase (predominantly localized upstream) and cytochrome P450 213 (predominantly localized downstream) that correlates with the distribution of these enzymes in vivo. The oxygen gradient chamber provides a means of probing the oxygen effects in vitro over a continuous range of O-2 tensions. In addition, this system serves as an in vitro model of zonation that could be further extended to study the role of gradients in ischemia-reperfusion injury, toxicity, and bioartificial liver design. (C) 2003 Wiley Periodicals, Inc.