The aim of this study was to investigate the mechanisms that might explain the observed route-dependent stereoselective pharmacokinetics (PK) of R/S-verapamil (R/S-VER) following oral and intravenous (iv) administration, by using a novel pig-specific physiologically based pharmacokinetic (PBPK) model suitable for investigations of first-pass extraction in the gut (E(G)) and the liver (E(H)). The PBPK model consisted of eight tissue compartments and was designed to simultaneously model the plasma concentration-time (PCT) profiles from three sampling sites after intrajejunal (ij) or iv administration of VER. The PBPK model successfully described the observed PCT profiles and E(H) over time for R- and S-VER. Extensive tissue binding to gut mucosa, liver, and lungs was an important determinant of the observed PK data. The stereoselective PK of VER was explained by a combination of several processes, including enantioselective plasma protein binding, blood-to-plasma partition, and gut mucosa and liver tissue distribution. The absence of stereoselectivity after iv dosing indicates that the first-pass tissue binding effect is an important factor in determining the steroselective PK of R/S-VER after oral administration. Additionally a combination of extensive liver tissue binding and a metabolite inhibition mechanism explained the time-dependent E(H) for both R- and S-VER. An in vitro-in vivo correlation of absorption needs to consider these processes because tissue binding may confound analysis of a drug's biopharmaceutical properties when using classical deconvolution or convolution techniques. In conclusion, a combination of PK data from multiple plasma sampling sites and a PBPK modeling approach provided a mechanistic understanding of processes involved in the intestinal absorption and first-pass extraction of R- and S-VER.