Without treatment, human African trypanosomiasis (HAT) is fatal. Drugs to treat the trypanosome infection within the CNS must cross the blood-brain and blood-cerebrospinal fluid barriers. However, the ability of existing anti-trypanosomal drugs to cross these barriers and reach targets within the CNS is unknown. Another consequence of the lack of resources put into combating HAT is the limited number of these drugs. These drugs are toxic and can encounter parasite resistance. Unfortunately, it is unlikely that new drugs will be available in the next decade. Consequently a clearer understanding on how these existing drugs enter and leave the CNS is essential if we are to optimise their use in terms of improving efficacy, reducing toxicity and in suggesting new combinations of drugs. In order to address this, we examined the CNS distribution of [³H]pentamidine isethionate by means of a brain/choroid plexus perfusion technique. In brief, adult BALB/c mice (25g) were anaesthetised (Fentanyl-Fluanisone-Midazolam 5mg/ml given at a rate of 10 ml/kg; i.p.) and the left ventricle of the heart cannulated. With the start of perfusion the right atrium was sectioned. The perfusion medium consisted of an oxygenated artificial plasma containing the radiolabelled drug and a vascular/extracellular space marker, [¹⁴C]sucrose. After a 20 min perfusion time the anaesthetised animal was decapitated and brain, choroid plexuses and plasma samples taken for radioactive counting. Integrity of the ³H-label to pentamidine in the arterial inflow was confirmed by HPLC (Poola et al., 2002). The results are tabulated and are [¹⁴C]sucrose corrected (mean ± S.E.M.; ml/100g). [³H]pentamidine was detected in the brain suggesting it can cross the blood-brain barrier, albeit to a limited extend. It can also accumulate in the choroid plexus cells. There was no significant difference (Student’s t-test; P>0.05) between [³H]pentamidine distribution in the absence and presence of unlabelled pentamidine, suggesting that a high affinity transporter is not involved in the CNS distribution of this drug.