The G-protein-coupled receptor 56 (GPR56) plays important roles in brain development and tumorigenesis. cDNA data suggest that GPR56 has potential to become a neural stem cell (NSC) or neural progenitor cell (NPC) marker. However, expression of GPR56 protein in human NSC/NPCs was not explored. Using specific antibodies and immunochemistry, we showed that GPR56 was highly expressed in nestin-positive NSC/NPCs in the ventricular/subventricular zone of human and mouse fetal brains, and in cultured neurospheres derived from both human and mouse fetal brains. Downregulation of GPR56 protein occurred earlier than that of nestin in differentiating neurosphere cultures. Loss of GPR56 protein was also evident in well-differentiated glial fibrillary acidic protein-positive astrocytes and betaIII-tubulin-positive neurons. Our data suggest that GPR56 can be used as an NSC/NPC marker within the neural cell lineage, especially in combination with nestin.