The Brn-3a POU family transcription factor is expressed only in posmitotic neurons in the central nervous system and identifies the first differentiated neurons to appear in the midbrain, hindbrain, and spinal cord during development. This factor is also induced when undifferentiated proliferating ND7 cells cease dividing and differentiate to a mature neuronal-like phenotype bearing numerous neurite processes. We show that overexpression of Brn-3a in undifferentiated ND7 cells induces a mature neuronal phenotype characterized by process outgrowth and the induction of genes encoding synaptic proteins, although the cells continue to proliferate. In contrast, the closely related factors Brn-3b and Brn-3c do not have this effect. Although the N-terminal activation domain of Brn-3a is required for maximum induction of neurite outgrowth and gene expression, these effects are primarily dependent on the DNA binding POU domain, which also acts as an activation domain. Overexpression of the isolated POU domain of Brn-3a is sufficient to induce neurite outgrowth, while the ability of full-length Brn-3a to do so is abolished by mutating a single amino acid in the Brn-3a POU homeodomain to its equivalent in Brn-3b. Thus, Brn-3a appears to play a critical role in the specification of the mature neuronal phenotype, acting by stimulating the expression of genes whose products are required for process outgrowth and synapse formation.