The aims of this study were to determine the effect of ketoconazole and rifampicin on the oral pharmacokinetics of ranitidine in human volunteers and to investigate the role of P-glycoprotein (P-gp) using in vitro systems. A randomized, placebo controlled crossover oral pharmacokinetic study was conducted in 12 healthy male human volunteers and in vitro (everted sac) and in situ (intestinal loop) studies were conducted in rats to study the role of P-gp. There was a statistically significant (p < 0.05) difference observed in the pharmacokinetic parameters C(max), AUC and MRT after pretreatment with rifampicin (600 mg orally once per day for 7 days). The C(max), AUC(0-infinity), and MRT were decreased by 53%, 52%, and 18%, respectively. Ketoconazole treatment (200 mg orally once per day for 5 days) increased the C(max), AUC(0-infinity) and T1/2 by 78%, 74%, and 56%, respectively, whereas T(max) was decreased by 31%. No statistically significant differences were observed in renal clearance (CLR) of ranitidine after treatment with either ketoconazole or rifampicin. Presence of ketoconazole significantly reduced the mean cumulative efflux concentrations (serosal to mucosal) of ranitidine to 35%, 41% and 55% in the duodenal, jejunum and ileal regions of the everted sacs, respectively, whereas, the mean cumulative efflux concentrations of ranitidine were increased by 14%, 36% and 25% in duodenal, jejunal and ileal regions of the rat small intestine, respectively, after pretreatment with rifampicin. The presence of ketoconazole improved the absorption of ranitidine significantly by increasing the percentage of total dose disappearing from the loops of duodenum, jejunum and ileum of rat small intestine by 82%, 84% and 85%, respectively. In contrast, treatment with rifampicin decreased the absorption of ranitidine by decreasing the percentage of total dose disappearing in duodenal, jejunal and ileal regions of the intestinal loops by 40%, 39% and 25%, respectively. Ranitidine was shown to be a P-gp substrate in vivo in human volunteers and it was found that oral bioavailability of ranitidine was influenced at the intestinal absorption phase.