We raised specific antibodies to the pleiotrophin and the midkine gene product, which comprise a novel heparin-binding growth factor family. Immunocytochemistry using these antibodies revealed that both proteins were strongly expressed in the developing cerebral cortex in the rat in distinct fashion. The midkine gene product was primarily detected in the embryonic period and was not observed in the postnatal cerebral cortex, whereas pleiotrophin was most intense in the early postnatal period, in addition to being present the embryonic period. Pleiotrophin was also localized in the adult cerebral cortex, albeit with much less intensity than in the embryonic and early postnatal period. These temporal patterns were consistent with those of the mRNA expression. The ultrastructural localization of both factors showed markedly similar profiles; immunopositive substances were primarily associated with the cell surface and were found preferentially in the regions where cell migration and neurite outgrowth take place, implying that this family functions primarily in developmental events mediated by cell-cell contact. The present results suggest that the midkine gene product and pleiotrophin are differentially involved in cortical development at different stages.