The mdr1-type P-glycoproteins can confer multidrug resistance to tumor cells by actively pumping a wide variety of drugs from the cell. To counteract this drug resistance, P-glycocoprotein-blocking agents are currently administered to patients during chemotherapy. However, this may also affect the normal physiological function(s) of the mdr1-type P-glycocoproteins. In order to establish these functions, we have generated mice with a genetic deficiency in both of their mdr1-type P-glycocoprotein genes. Our results indicate the mdr1-type P-glycocoproteins are not essential for basic physiological functions. However, mice without mdr1-type P-glycocoproteins display drastic alterations in the pharmacological handling of drugs, demonstrating an important role for mdr1-type P-glycocoprotein in the blood-brain barrier, where it prevents the accumulation of many drugs in the brain. Moreover, we found that intestinal P-glycocoprotein has a prominent role in the extrusion of several drugs from the blood into the intestinal lumen, and in preventing drugs in the intestinal lumen from (re-)entering the bloodstream. The latter property can have important implications for the oral bioavailability of many drugs. Our results indicate that effective P-glycocoprotein-blocking agents should be used with caution, given the potentially extensive pharmacokinetic effects of treatment with these compounds.