The objective of present investigation was to study the effect of verapamil on the pharmacokinetics of irinotecan in order to evaluate the role of P-glycoprotein (P-gp) in irinotecan disposition. An in vitro study using Caco-2 intestinal cell monolayer was first carried out to determine the effect of verapamil on the function of intestinal P-gp. Verapamil (25mg/kg) was administered orally 2h before irinotecan oral (80 mg/kg) or intravenous (20mg/kg) dosing in female Wistar rats. Plasma and biliary samples were collected at specified time points from control and treated animals to determine irinotecan and its metabolite, SN-38 concentrations. Bi-directional transport and inhibition studies in Caco-2 cells indicated irinotecan to be a P-gp substrate and the function of intestinal P-gp was significantly inhibited in presence of verapamil. After oral irinotecan dosing, the mean area under the plasma concentration-time curve (AUC) was found to be 14.03+/-2.18 microgh/ml which was increased significantly, i.e. 61.71+/-15.0 microgh/ml when verapamil was co-administered (P<0.05). Similarly, the mean maximum plasma concentration of irinotecan increased from 2.93+/-0.37 microg/ml (without verapamil) to 10.75+/-1.0 microg/ml (with verapamil) (P<0.05). There was approximately 4-5-folds increase in apparent bioavailability. On the other hand, the intravenous irinotecan administration with verapamil resulted in small but statistically significant effect on AUC (10.76+/-2.0 to 23.3+/-3.8 microgh/ml; P<0.05) and systemic clearance (1206.4+/-159.7 to 713.5+/-78.2 ml/(hkg)). In addition, SN-38 showed significant change in oral pharmacokinetic parameters and minor changes in intravenous pharmacokinetic profile. Biliary excretion curves of both irinotecan and SN-38 were lowered by verapamil. The mean percent of irinotecan excreted into bile over 5h following intravenous and oral administration was found to be 8% and 1%, respectively, which was further reduced to half when treated with verapamil. These results are quite stimulating for further development of a clinically useful oral formulation of irinotecan based on P-gp inhibition.