In recent studies we have demonstrated that glycogen synthase kinase 3beta (GSK3beta) and its substrate microtubule-associated protein 1B (MAP1B) regulate the microtubule cytoskeleton during axon outgrowth. To further examine the role GSK3beta plays in axon outgrowth we investigated the expression of GSK3beta and its activity towards MAP1B during nerve growth factor (NGF)-stimulated PC12 cell differentiation. Levels of GSK3beta expression increase relatively little during the course of differentiation. However, the expression of a novel GSK3beta isoform characterised by a reduced mobility on SDS gels is induced by NGF. Expression of this isoform and the GSK3beta-phosphorylated isoform of MAP1B (MAP1B-P) are induced in parallel in response to NGF. This increase lags behind initial neurite formation and the expression of MAP1B in these cells by about two days and coincides with a period when the majority of cells are extending existing neurites. MAP1B and GSK3beta are expressed throughout the PC12 cell but MAP1B-P expression is restricted to the growth cones and neurites. Consistent with these observations, we find that neurite extension is more sensitive to the GSK3 inhibitor Li+ than neurite formation and that this correlates with an inhibition of MAP1B phosphorylation. Additionally, GSK3beta from PC12 cells not exposed to NGF can not phosphorylate MAP1B in vitro. However, a soluble factor in differentiated PC12 cell extracts depleted of GSK3beta can activate MAP1B phosphorylation from undifferentiated cell extracts otherwise devoid of kinase activity. These experiments provide evidence for an NGF-mediated regulation of MAP1B phosphorylation in growing neurites by the induction of a novel isoform of GSK3beta.