Prediction of clearance in drug discovery currently relies on human primary hepatocytes, which can vary widely in drug-metabolizing enzyme activity. Potential alternative in vitro models include the HepaRG cell (from immortalized hepatoma cells), which in culture can express drug-metabolizing enzymes to an extent comparable to that of primary hepatocytes. Utility of the HepaRG cell will depend on robust performance, relative to that of primary hepatocytes, in routine high-throughput analysis. In this study, we compared intrinsic clearance (CL int) in the recently developed cryopreserved HepaRG cell system with CL int in human cryopreserved pooled hepatocytes and with CL int in vivo for 26 cytochrome P450 substrate drugs. There was quantitative agreement between CL intin HepaRG cells and human hepatocytes, which was linear throughout the range of CL int (1-2000 ml · min -1 · kg -1) and not dependent on particular cytochrome P450 involvement. Prediction of CL int in HepaRG cells was on average within 2-fold of in vivo CL int (using the well stirred liver model), but average fold error was clearance-dependent with greater underprediction (up to at least 5-fold) for the more highly cleared drugs. Recent reporting of this phenomenon in human hepatocytes was therefore confirmed with the hepatocytes used in this study, and hence the HepaRG cell system appears to share an apparently general tendency of clearance- limited CL int in cell models. This study shows the cryopreserved HepaRG cell system to be quantitatively comparable to human hepatocytes for prediction of clearance of drug cytochrome P450 substrates and to represent a promising alternative in vitro tool. Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics.