Multidrug transporters of the ATP-binding cassette family play an important role in regulating drug distribution and efflux in the brain, owing to their selective distribution in microvessels and choroid plexus. Their expression pattern and cellular distribution remain controversial, in part due to technical difficulties in localizing these membrane proteins in closely associated cells, such as endothelial cells and astrocytic end-feet at the blood-brain barrier. Here, we used high-resolution immunofluorescence staining with cell-type specific markers to investigate the distribution of major ATP-binding cassette transporters in mouse brain. We report that four ATP-binding cassette transporters, Mdr1, Mrp1, Mrp2 and Mrp5 can be detected in brain endothelial cells, forming three distinct layers, with Mdr1 and Mrp5 being located on the luminal side, Mrp1 on the abluminal (basal) side, and Mrp2 in between. Mrp3 and Mdr3 were undetectable. In choroid plexus, only Mrp1, Mrp2 and Mrp3 were detected, again with a differential distribution. Mrp1 was targeted basolaterally in epithelial cells, Mrp2 was restricted to endothelial cells, and Mrp3 was co-localized with zonula occludens-1 at tight junctions. Analysis of Mdr1a(0/0) and Mrp1(0/0) mice, generated in the FVB strain, revealed no major alteration in expression of the remaining transporters. An unexpected strain difference was unraveled, with wildtype FVB mice selectively lacking Mrp2 protein in brain, but not liver. In conclusion, these results indicate that ATP-binding cassette transporters provide multiple penetration barriers in the blood-brain barrier and choroid plexus, with a selective cellular and subcellular distribution, emphasizing their potential role for drug resistance in neurological disorders, such as epilepsy.