Mdr65^KG08723 mutant flies show high relative brain-specific accumulation of Rhodamine B dye, suggesting that Mdr65 promotes efflux of xenobiotics from the brain. Whole animal controls show that dosing and elimination of Rhodamine B are not affected by the Mdr65^KG08723 mutation. Brain accumulation of 3kDa FITC-dextran remains near background levels in both wild-type and Mdr65^KG08723 animals, indicating that the P{SUPor-P}Mdr65^KG08723 insertion does not significantly disturb the paracellular diffusion barrier. The blood-brain barrier in Mdr65^KG08723 mutants is indistinguishable from wild-type relative to all highly-charged small molecules tested. Brain levels of Rhodamine B are similar between Mdr65^KG08723 and wild-type flies at early time points, showing that the P{SUPor-P}Mdr65^KG08723 insertion does not affect initial brain penetration by Rhodamine B. At 4, 8, and 16 hours after injection, however, Mdr65^KG08723 mutant flies exhibit significantly higher brain levels of Rhodamine B than wild-type flies, suggesting the mutant flies have a markedly reduced Rhodamine B efflux from the brain.