Studies of drug distribution to brain should be controlled for the experimental method used. Numerous methods have been employed to ascertain brain distribution and many of these approaches use anesthetic agents. The in situ rat brain perfusion method is one of the most sensitive and widely used methods for evaluating brain distribution profiles. There has been no evaluation of the effects of anesthetic agents on parameters associated with this method (i.e. cerebral perfusion fluid flow, brain vascular volume and blood-brain barrier permeability). We evaluated the effects of the anesthetic agents pentobarbital and ketamine combinations on these baseline parameters. The results suggest that the anesthetic agent has no effect on these parameters and anesthetic selection is open to the choice of the investigator when using the perfusion method.