All-trans retinoic acid (atRA), the active metabolite of vitamin A, is a ligand for several nuclear receptors and acts as a critical regulator of many physiologic processes. The cytochrome P450 family 26 (CYP26) enzymes are responsible for atRA clearance, and are potential drug targets to increase concentrations of endogenous atRA in a tissue-specific manner. Talarozole is a potent inhibitor of CYP26A1 and CYP26B1, and has shown some success in clinical trials. However, it is not known what magnitude of change is needed in tissue atRA concentrations to promote atRA signaling changes. The aim of this study was to quantify the increase in endogenous atRA concentrations necessary to alter atRA signaling in target organs, and to establish the relationship between CYP26 inhibition and altered atRA concentrations in tissues. Following a single 2.5-mg/kg dose of talarozole to mice, atRA concentrations increased up to 5.7-, 2.7-, and 2.5-fold in serum, liver, and testis, respectively, resulting in induction of Cyp26a1 in the liver and testis and Rar β and Pgc 1β in liver. The increase in atRA concentrations was well predicted from talarozole pharmacokinetics and in vitro data of CYP26 inhibition. After multiple doses of talarozole, a significant increase in atRA concentrations was observed in serum but not in liver or testis. This lack of increase in atRA concentrations correlated with an increase in CYP26A1 expression in the liver. The increased atRA concentrations in serum without a change in liver suggest that CYP26B1 in extrahepatic sites plays a key role in regulating systemic atRA exposure.