The novel gastroprokinetic agent acotiamide improves gastric motility by inhibiting acetylcholinesterase activity in stomach; however, the mechanism of distribution of acotiamide from blood to stomach has not been clarified. Here, the tissue distribution of acotiamide was investigated in rats. The tissue-to-plasma concentration ratio (Kp,app,in vivo) for stomach decreased from 4.1 to 2.4 mL/g of tissue at steady state with increasing plasma concentrations, whereas the Kp,app,in vivo for skeletal muscle was much lower and constant, regardless of the concentration of acotiamide in plasma. In vitro binding to stomach tissue protein exhibited a linear profile, with a predicted K p,app,in vitro of 2.2 from free fractions under linear conditions. Therefore, protein binding to stomach tissue might only play a limited role in the stomach distribution of acotiamide. The influx permeability (f u,b ร PS inf,app ) in the stomach exhibited dose-dependent saturation at the lowest range of examined blood unbound concentrations of acotiamide, whereas that in skeletal muscle exhibited only minimal dose dependence. In addition, the unbound concentration ratio of stomach to plasma (2.8) at steady state was markedly higher than unity. Taken together, these results suggest that carrier-mediated concentrative uptake processes play an important role in the distribution of acotiamide to the stomach but not skeletal muscle.