Kinetic parameters describing hepatic uptake in hepatocytes are frequently estimated without appropriate incorporation of bidirectional passive diffusion, intracellular binding, and metabolism. A mechanistic two-compartment model was developed to describe all of the processes occurring during the in vitro uptake experiments performed in freshly isolated rat hepatocytes plated for 2 h. Uptake of rosuvastatin, pravastatin, pitavastatin, valsartan, bosentan, telmisartan, and repaglinide was investigated over a 0.1 to 300 μM concentration range at 37°C for 2 or 45-90 min; nonspecific binding was taken into account. All concentration-time points were analyzed simultaneously by using a mechanistic two-compartment model describing uptake kinetics [unbound affinity constant (K m,u), maximum uptake rate (V max), unbound active uptake clearance (CL active,u)], passive diffusion [unbound passive diffusion clearance (P diff,u)], and intracellular binding [intracellular unbound fraction (fu cell)]. When required (telmisartan and repaglinide), the model was extended to account for the metabolism [unbound metabolic clearance (CL met,u)]. The CL active,u ranged 8-fold, reflecting a 11-fold range in uptake K m,u, with telmisartan and valsartan showing the highest affinity for uptake transporters (K m,u 70% of total uptake for all drugs investigated and was 4- and 112-fold greater than CL met,u for telmisartan and repaglinide, respectively. Modeling of uptake kinetics in conjunction with metabolism improved the precision of the uptake parameter estimates for repaglinide and telmisartan. Recommendations are made for uptake experimental design and modeling strategies. Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics.