Drug depletion-time profiles in isolated hepatocytes, as well as microsomes, have become a standard method of assessing hepatic metabolic clearance in vitro. There is a previously described adaptation of the depletion approach to allow determination of hepatic uptake by transporters in addition to metabolism (Drug Metab Dispos 35:859-865, 2007). Dual incubations are performed where one set of incubations undergo conventional methodology, whereas for the second set, cells and media are separated for determination of drug loss from the media. The utility of this dual incubation approach has been assessed using eight drugs (atorvastatin, clarithromycin, erythromycin, fexofenadine, pitavastatin, repaglinide, rosuvastatin, and saquinavir) with a range of active uptake, passive permeability, cell binding, and metabolic characteristics. Four of these compounds (fexofenadine, rosuvastatin, pitavastatin, and atorvastatin) show a biphasic time profile when assessing drug loss from media indicative of hepatic uptake before elimination within the hepatocyte, which is distinct from the time profile in a conventional incubation, and show higher clearances. The four other compounds (clarithromycin, saquinavir, erythromycin, and repaglinide) show identical depletion-time profiles (and clearances) in both sets of incubations. Whether or not the biphasic nature (and higher clearance) is evident, indicating transporter activity for a particular drug, appears to be dependent on its passive permeability. Using the parameter Kpu to reflect the relative importance of hepatic transporters versus passive diffusion, a value of 10 was identified as a cutoff for whether the biphasic nature was evident; those compounds in excess of 10 show this characteristic clearly. There appears to be no relationship between the presence of the biphasic nature and any other parameter, including cellular binding, extent of metabolism, or the magnitude of active uptake. Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics.