The in vivo effects of berberine (BBR), the widely used bioactive herbal ingredient from many traditional Chinese medicinal herbs, on the pharmacokinetics of carbamazepine (CBZ, a substrate of CYP3A) and its metabolite carbamazepine 10,11-epoxide (ECBZ), digoxin (DIG, a substrate of P-gp) and cyclosporine A (CsA, a dual substrate of CYP3A and P-gp) were evaluated in rats. After a 2-week pretreatment with BBR, the pharmacokinetic parameters of i.g. administered CBZ and ECBZ were not significantly altered. The pharmacokinetics of i.v. administered DIG was not modified by single and 2-week pretreatments with BBR, but a dose-dependent increase in AUC and C(max) was observed in the i.g. administered DIG parameters in rats. The AUCs of DIG with BBR (30 mg/kg, 100 mg/kg) were 133%, 170% (single) and 123%, 169% (2-week) of control, respectively. The AUC and C(max) of i.g. administered CsA with a 2-week pretreatment with BBR increased by 62% and 43% (BBR 30 mg/kg, p < 0.05), 96% and 60% (BBR 100 mg/kg, p < 0.01), compared with the control. In conclusion, berberine produced a dose-dependent increased bioavailability of digoxin and cyclosporine A by inhibition of intestinal P-gp. No significant changes in CYP3A activity by berberine were observed.