Pentamidine is used to treat the haemolymphatic stage of human African trypanosomiasis (HAT), a fatal illness if untreated. Pentamidine is not thought to enter the brain sufficiently due to extrusion by efflux transporters at the blood-brain barrier (BBB) (Sanderson, Dogruel et al. 2009). It is hypothesized that inhibition of the CNS efflux transporters will lead to increased pentamidine entry into the brain and thus provide an alternative treatment for the neurological stage of HAT. However, to determine the exact identity of the uptake and efflux transporters of pentamidine at the human and mouse BBB, further study is required. To investigate this, human (hCMEC/D3) and mouse (bEND.3) brain endothelial cell lines were employed. Accumulation assays were carried out as described by Watson, Dogruel et al. (2012) using 3H Pentamidine and 14C Sucrose as a vascular space marker (n= 3 plates, 6 wells per treatment). Western blots and immunofluorescence confirmed the presence of P-glycoprotein and organic cation transporter 1 (OCT1). All data was analysed by two-way ANOVA using Sigmaplot software. The data suggests that there is an uptake transporter for pentamidine in both cell lines. This could potentially be the OCT system since haloperidol metabolites have been shown to inhibit this system (Kang, Lee et al. 2006). The role of the OCT system is further highlighted as the presence of 500µM amantadine significantly reduced 3H pentamidine accumulation. The increase in accumulation of 3H pentamidine under ATP depletion suggests that ATP-dependent efflux transporters (P-glycoprotein, breast-cancer resistance protein, multi-drug resistance protein) are involved in the efflux of this drug into the blood. In conclusion, multiple transporters are involved in the distribution of pentamidine across mouse and human cerebral capillary endothelial cells. In addition, there are significant differences in the response of the human and mouse cells invitro to pentamidine.