Spectrin is an actin-binding membrane skeleton protein involved in the maintenance of cell shape and generation of distinct membrane protein domains. Actin binds to the N-terminal domain of beta-spectrin. To examine the function of spectrin-actin interaction in neurons, we sought to disrupt this interaction in differentiating NB 2a neuroblastoma cells by microinjecting an N-terminal domain-specific anti-beta-spectrin antibody. We found that microinjection of the affinity-purified N-terminal domain-specific anti-beta-spectrin inhibited the extension of the neurites in NB 2a/dl cells. The microinjected cells remained flat, and put out many filopodia-like processes; but these processes failed to extend when the cells were induced to differentiate in the presence of dbc AMP or in serum-free medium. The N-terminal domain-specific anti-beta-spectrin also inhibited the binding of spectrin to actin. By contrast, the microinjection of monospecific anti-alpha-spectrin(G) did not inhibit neurite extension. These results suggest that beta-spectrin-actin interaction may be required for neurite extension, which is critical for development of polarity in nerve cells.