Predictions of intrinsic clearance (CL(int)) from human liver microsomes often underestimate in vivo observations. In this study, a series of five benzodiazepines was used as prototypic CYP3A4 substrates to investigate the prediction of clearance from the less studied alternative in vitro system, cryopreserved human hepatocytes. Formation of the two major metabolites from midazolam, triazolam, diazepam, flunitrazepam, and alprazolam was measured in cryopreserved human hepatocytes from five donors; the kinetics were characterized and CL(int) values were determined and scaled to predict CL(int) in vivo. At least one of the two major pathways of metabolic clearance of each benzodiazepine was characterized by autoactivation in hepatocytes; the extent to which this occurred varied depending on substrate and liver (up to 8-fold). Heteroactivation by testosterone of these pathways was also observed (up to 6-fold). The maximum autoactivated clearance was used to predict in vivo CL(int) (1.6-138 ml/min/kg) which closely agreed with values previously obtained using human liver microsomes. Comparison with in vivo CL(int) indicates that cryopreserved human hepatocytes systematically underpredict CL(int). When three previous studies (documenting CL(int) for substrates of various enzymes in cryopreserved human hepatocytes using drug depletion-time profiles) were considered as well, the combined data show a consistent underprediction of 5.6-fold. Collectively it is demonstrated that the predicted hepatic intrinsic clearances from cryopreserved hepatocytes show an excellent rank order with in vivo findings but are systematically underpredicting the in vivo value.