Molecular mechanisms of endothelial cell differentiation during blood-brain barrier (BBB) development is not well understood due to the lack of specific molecular markers. Here we describe that expression of the mouse multidrug resistance 1a/3 (mdr1a/3) gene can be detected specifically in subsets of vascular endothelial cells associated with neural tissues at as early as embryonic day 10.5 (E10.5). This onset of mdr1a/3 gene expression coincides with the previously described first appearance of morphologically distinct endothelial cells in neural tissues during BBB development. To our knowledge, the mdr1a/3 gene is the earliest endothelial cell differentiation marker gene during BBB development described thus far. In addition, we have found that neither the level nor pattern of mdr1a/3 gene expression in BBB endothelial cells is affected by aberrant cortical neuronal layers in mutant mouse reeler.