Nogo-A is a transmembrane protein originally discovered in myelin, produced by postnatal CNS oligodendrocytes. Nogo-A induces growth cone collapse and inhibition of axonal growth in the injured adult CNS. In the intact CNS, Nogo-A functions as a negative regulator of growth and plasticity. Nogo-A is also expressed by certain neurons. Neuronal Nogo-A depresses long-term potentiation in the hippocampus and modulates neurite adhesion and fasciculation during development in mice. Here we show that Nogo-A is present in neurons derived from human midbrain (Lund human mesencephalic (LUHMES) cell line), as well as in embryonic and postnatal mouse midbrain (dopaminergic) neurons. In LUHMES cells, Nogo-A was upregulated threefold upon differentiation and neurite extension. Nogo-A was localized intracellularly in differentiated LUHMES cells. Cultured midbrain (dopaminergic) neurons from Nogo-A knock-out mice exhibited decreased numbers of neurites and branches when compared with neurons from wild-type (WT) mice. However, this phenotype was not observed when the cultures from WT mice were treated with an antibody neutralizing plasma membrane Nogo-A. In vivo, neither the regeneration of nigrostriatal tyrosine hydroxylase fibers, nor the survival of nigral dopaminergic neurons after partial 6-hydroxydopamine lesions was affected by Nogo-A deletion. These results indicate that during maturation of cultured midbrain (dopaminergic) neurons, intracellular Nogo-A supports neurite growth initiation and branch formation.