We determined the effect of zosuquidar.3HCl, an inhibitor of P-gp, on the penetration of the anticancer drug paclitaxel into the brain. Zosuquidar.3HCl was administered orally at 25 and 80 mg/kg 1 h before i.v. paclitaxel and i.v. at 20 mg/kg 10 min and 1 h before paclitaxel. The concentrations of paclitaxel in plasma and tissues and of zosuquidar.3HCl in plasma were quantified by high-performance liquid chromatography. The results revealed 3.5-fold and 5-fold higher paclitaxel levels in the brain of wild-type mice treated orally with 25 and 80 mg/kg zosuquidar.3HCl, respectively. However, complete inhibition as in P-gp knockout mice (11-fold increase) was not achieved. Zosuquidar.3HCl also increased the paclitaxel concentrations in plasma and tissues to levels similar to those observed in P-gp knockout mice, suggesting selective P-gp inhibition of zosuquidar.3HCl. When zosuquidar.3HCl was administered i.v. 10 min before paclitaxel, the paclitaxel levels in the brain of wild-type mice increased by 5.6-fold, whereas the increase was only 2.1-fold when zosuquidar.3HCl was administered 1 h before paclitaxel. This suggests that the inhibition of P-gp at the blood-brain barrier by zosuquidar.3HCl is rapidly reversible and that the concentrations of zosuquidar.3HCl in the plasma have already declined to levels insufficient to inhibit P-gp at the blood-brain barrier. In conclusion, zosuquidar.3HCl is only moderately active as an inhibitor of P-gp at the blood-brain barrier.