Morphologically polarized hippocampal neurons, grown in culture for two days, contain immunoreactivity of the growth-associated protein B-50 along the plasma membrane of both dendrites and axons. In mature hippocampal neurons, both in vitro and in vivo, B-50 is located in the axon. In order to assess at which stage during neuronal differentiation B-50 is selectively located in the axon, an immuno-light and electron-microscopic study was performed on rat hippocampal neurons developing in vitro. B-50 immunofluorescence was detected in the axon, dendrites and soma of two-day-old polarized neurons. Simultaneously, microtubule-associated protein 2, a marker specific to dendritic microtubules, was predominantly found in the soma, the short dendritic processes and at the base of axonal growth cones. In hippocampal neurons cultured beyond seven days in vitro, microtubule-associated protein 2 immunofluorescence is restricted to the cell soma and dendrites. The spatial distribution of B-50, however, varies. In solitary neurons maturing without interneuronal contacts, B-50 immunofluorescence is observed in axons and in the dendrosomatic domain characterized by the presence of microtubule-associated protein 2. In contrast, in high-density cell cultures B-50 immunofluorescence is absent in the cell body and dendrites, but punctate in axons running along the dendrites. Electron microscopy was carried out on hippocampal neurons of eight to 21 days in vitro to study the process of redistribution of B-50 at the subcellular level. In neurons of eight days in vitro with prominent synapses, B-50 immunoreactivity is significantly elevated at the axonal plasma membrane compared to the plasma membrane of the dendrites and the soma. In neurons from the same culture without synapses, B-50 immunoreactivity is distributed rather densely along the plasma membrane of the soma, dendrites, and on the axonal plasma membrane. A similar B-50 distribution is observed in mature neurons cultured at low cell density without interneuronal cell contacts, for 15 days in vitro. In high-density cell cultures of 21 days in vitro, B-50 is virtually absent at the plasma membrane of the soma and dendrites, and heterogenously distributed along the plasma membrane of axon and axonal varicosities. Our results indicate that selective sorting of B-50 into axons occurs after initial morphological polarization of hippocampal neurons and is correlated with the formation of synapses and with the cessation of dendritic outgrowth.