The biotin derivative, N-(2-aminoethyl) biotinamide hydrochloride, or Neurobiotin, has been shown recently to be a useful marker for intracellular and anterograde tracing. The properties of Neurobiotin as a tracer were further examined in this study by making pressure injections into different regions of the cerebral cortex or the rostral neostriatum of rats or by incubating striatal cells in culture with the tracer. Results showed extensive anterograde transport of Neurobiotin in cortical axons and terminals within the neostriatum 2-70 h after single or multiple cortical injections of the tracer. Similarly, profuse axonal projections to the medial portion of the globus pallidus were seen after an injection of Neurobiotin into the rostral neostriatum. Transneuronal labeling of medium-size neostriatal neurons was observed following injections of Neurobiotin into the prefrontal cortex. At the ultrastructural level, anterogradely labeled cortical axon terminals and transneuronally labeled neurons were readily identified in the caudate-putamen by the presence of both fine particulate and large punctate reaction products. Retrograde fillings of neurons resembling a Golgi-impregnation were seen in the ventral posterior complex of the thalamus after injections in the sensorimotor cortex. Neurons in the medial globus pallidus were also retrogradely labeled following tracer injections in the rostral caudate-putamen. Finally, Neurobiotin was readily and selectively taken up by striatal neurons in culture, where it extensively labeled somata and neurites. These results show that Neurobiotin is a versatile new tracer, which can be potentially useful for the study of neuronal organization in vivo and in vitro.