The transcription factor NGFI-A is an early response gene that has been implicated in the regulation of cell growth and differentiation and, more recently, in apoptosis. This gene is expressed in many tissues, and is very abundant in the brain. However, little is known about its functional role in the differentiation of this tissue. In the present work we investigated the role of NGFI-A in serum withdrawal-induced differentiation in N2A neuroblastoma cells. To do so, we studied the effect of NGFI-A antisense oligonucleotides and NGFI-A overexpression on this process. We show that neuroblastoma cells treated with an NGFI-A antisense oligonucleotide do not undergo normal morphological differentiation after serum withdrawal, whereas N2A cells overexpressing this gene extend long neurites, even in the presence of serum. We also show that NGFI-A overexpression is accompanied by an increase in the amount of phosphorylated microtubule-associated protein MAP1B, which has been associated with neurite outgrowth. Our results suggest that the NGFI-A gene plays an important role in neurite extension.