The EFA6 family is a member of guanine nucleotide exchange factors (GEFs) that can activate ARF6 specifically in vitro. In this study, we determined the complete primary sequence of mouse EFA6D encoding a protein of 1004 amino acids with a calculated molecular weight of 111,207 Da. In ARF pull-down assay, EFA6D showed a preferential GEF activity toward ARF6. RT-PCR analysis revealed the widespread tissue distribution of EFA6D and the high expression of EFA6A, C and D in the brain. In situ hybridization analysis demonstrated a distinct spatiotemporal expression pattern of EFA6D from those of EFA6A and C in mouse brain. Furthermore, immunoblot analysis revealed that EFA6D was highly concentrated in the postsynaptic density fraction. These findings suggest differential spatiotemporal regulation of ARF6 by three members of the EFA6 family in the brain.