This study was conducted to examine the oral bioavailability and the possibility of enterohepatic recirculation of otilonium bromide in rats. A sensitive LC/MS/MS assay (LLOQ 0.5 ng/mL) was developed for the determination of otilonium and applied to i.v. and oral administration studies in bile duct cannulated (BDC) and non-BDC rats. After i.v. injection to BDC rats (1 mg/kg as otilonium), average $t_{1/2}$, CL, $V_z$ and AUC were $7.9;{pm};1.9;h,;8.7;{pm};3.1$ mL/min/kg, $5.7;{pm};1.4$ L/kg and $2,088;{pm};676;ng{cdot}h/mL$, respectively, and these values were comparable to those found in non-BDC rats. The percentages of i.v. dose excreted unchanged in bile and urine in BDC rats were $11.6;{pm};3.0$ and $3.1;{pm};0.7$%, respectively. Upon oral administration to non-BDC rats (20 mg/kg as otilonium), $t_{1/2},;C_{max},; T_{max}$ and AUC were $6.4;{pm};1.3;h,;182.8;{pm};44.6;ng/mL,; 1.9;{pm};1.6;h$ and $579;{pm};113;ng{cdot}h/mL$, respectively. The absolute oral bioavailability was low (1.1%), while the drug was preferentially distributed to gastrointestinal tissues. A secondary peak was observed in the serum concentration-time profiles in non-BDC rats following both i.v. and oral administration, indicating that otilonium bromide was subject to enterohepatic recirculation.