Species differences occur in the brain concentrations of drugs, but the reasons for these differences are not yet apparent. This study was designed to compare brain uptake of three radiolabeled P-glycoprotein (P-gp) substrates across species using positron emission tomography. Brain concentrations and brain-to-plasma ratios were compared; [(11)C]verapamil in rats, guinea pigs, and monkeys; [(11)C](S)-(2-methoxy-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethylamino)-2(S)-phenylpiperidine (GR205171) in rats, guinea pigs, monkeys, and humans; and [(18)F]altanserin in rats, minipigs, and humans. The fraction of the unbound radioligand in plasma was studied along with its metabolism. The effect of P-gp inhibition was investigated by administering cyclosporin A (CsA). Pronounced species differences were found in the brain and brain-to-plasma concentrations of [(11)C]verapamil, [(11)C]GR205171, and [(18)F]altanserin with higher brain distribution in humans, monkeys, and minipigs than in rats and guinea pigs. For example, the brain-to-plasma ratio of [(11)C]GR205171 was almost 9-fold higher in humans compared with rats. The species differences were still present after P-gp inhibition, although the increase in brain concentrations after P-gp inhibition was somewhat greater in rats than in the other species. Differences in plasma protein binding and metabolism did not explain the species-related differences. The findings are important for interpretation of brain drug delivery when extrapolating preclinical data to humans. Compounds found to be P-gp substrates in rodents are likely to also be substrates in higher species, but sufficient blood-brain barrier permeability may be retained in humans to allow the compound to act at intracerebral targets.