The mammalian RYK is an orphan receptor that contains a catalytically inactive tyrosine-kinase-related domain. Its Drosophila homolog, Lio/Drl, is required for axon pathfinding in developing brain. Our previous study suggested that RYK mRNA is expressed in nestin-positive progenitor cells and neurons. In the present study, immunohistochemistry has been used to further localize RYK in the central nervous system of rats to identify the lineage of the RYK-expressing cells. In the embryonic forebrain, RYK colocalized with nestin in the ventricular zone and with MAP2 in the cortical plate, suggesting that RYK is expressed in neural progenitor cells and neurons. Localization of RYK in embryonic spinal cord also suggested its expression in both cell types. In primary cultures of rat cerebrum, RYK expression was observed in all neurons, as well as in a significant population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes. However, no RYK expression was detected in type-1 astrocytes or microglia. Multipotent neural stem cell line MNS-70 was also analyzed for expression of RYK, and most of the cells were positive for both RYK and nestin in the undifferentiated stage. In the differentiated stage, expression of RYK was detected in the neurons, but not in type-1 astrocytes. In conclusion, RYK is expressed in nestin-positive progenitor cells and neurons, and in a certain population of oligodendrocytes, O-2A progenitor cells, and type-2 astrocytes in developing CNS. These findings show that expression of RYK in rat CNS is tightly regulated in a cell-type-specific manner.