Prediction of clearance - a vital component of drug discovery - remains in need of improvement and, in particular, requires more incisive assessment of mechanistic methodology in vitro, according to a number of recent reports. Although isolated hepatocytes have become an irreplaceable standard system for measurement of intrinsic hepatic clearance mediated by active uptake transport and metabolism, lack of prediction reliability appears to reflect a lack of methodological validation, especially for highly cleared drugs, as we have previously shown. Here, novel approaches were employed to explore fundamental experimental processes and associated potential limitations of in vitro predictions of clearance. Rat hepatocytes deemed non-viable by trypan blue staining showed undiminished metabolic activity for probe P450 substrates midazolam and propranolol; supplementation with NADPH enhanced these activities. Extensive permeabilisation of the plasma membrane using saponin showed either full or minimal P450 activity depending on the presence or absence of 1 mM NADPH, respectively. Shaking of incubations facilitated P450 metabolic rates up to 5-fold greater than static incubation, depending on intrinsic clearance, indicating the critical influence of the unstirred water layer (UWL). Permeabilisation allowed static incubation metabolic rates to approach those of shaking for intact cells, indicating an artificially induced breakdown of the UWL. Permeabilisation combined with shaking allowed an increased metabolic rate for saquinavir, resolving the membrane permeability limitation for this drug. These findings advance the interpretation of the rate limiting processes involved in intrinsic clearance measurements, and could be critical for successful in vitro prediction.