Midkine (MK) is the product of a retinoic acid responsive gene, MK, and is the initial member of a new family of heparin binding factors. Recombinant MK produced by an L-cell clone transfected with an MK cDNA was purified to homogeneity. When rat embryonic brain cells were cultured in a serum-free medium on plastic dishes coated with purified MK, a number of neurites extended, formed networks and fasciculated. The majority of neurons continued to extend neurites for a week. In control cultures, neurite extension was observed only in a few cells. Neurotrophic activity of retinoic acid may be mediated by MK. MK had weak but significant mitogenic activity to 3T3 fibroblasts but not to NRK cells. Thus, MK and other members of the family share a part of the activities.