Many therapeutic drugs are excluded from entering the brain, due to their lack of transport through the blood-brain barrier (BBB). To overcome this problem, we have developed a novel method in which short, naturally derived peptides (16-18 amino acids) cross the cellular membranes of the BBB with high efficiency and without compromising its integrity. The antineoplastic agent doxorubicin (dox) was coupled covalently to two peptides, D-penetratin and SynB1. The ability of dox to cross the BBB was studied using an in situ rat brain perfusion technique and also by i.v. injection in mice. In the brain perfusion studies, we first confirmed the very low brain uptake of free radiolabeled dox because of the efflux activity of P-glycoprotein at the BBB. By contrast, we have demonstrated that when dox is coupled to either the D-penetratin or SynB1 vectors, its uptake was increased by a factor of 6, suggesting that the vectorized dox bypasses P-glycoprotein. Moreover, using a capillary depletion method, we have shown that vectorization of dox led to a 20-fold increase in the amount of dox transported into brain parenchyma. Intravenous administration of vectorized dox at a dose of 2.5 mg/kg in mice led to a significant increase in brain dox concentrations during the first 30 min of postadministration, compared with free dox. Additionally, vectorization led to a significant decrease of dox concentrations in the heart. In summary, our results establish that the two peptide vectors used in this study enhance the delivery of dox across the BBB.