Microtubule-associated protein 1B, formerly also known as microtubule-associated protein 5, is the first structural microtubule accessory protein to appear in outgrowing axons. In PC12 pheochromocytoma cells microtubule-associated protein 1B levels increase several-fold after the addition of nerve growth factor and this increase is correlated with the initiation of process formation. To determine whether microtubule-associated protein 1B is essential for neurite outgrowth, we used antisense oligodeoxynucleotides to inhibit its expression in nerve growth factor-treated PC12 cells in the rat. The application of several different antisense oligodeoxynucleotides to the microtubule-associated protein 1B mRNA sequence inhibited both microtubule-associated protein 1B expression and neurite extension. Specificity was shown by the lack of effect of control sense oligonucleotides and by the lack of effect of the microtubule-associated protein 1B antisense oligodeoxynucleotides on the expression of either tubulin or microtubule-associated protein 3, another microtubule-associated protein whose synthesis is stimulated by nerve growth factor treatment of PC12 cells. After removal of the antisense oligodeoxynucleotides, microtubule-associated protein 1B expression recovered to normal levels and the cells grew normal neurites with the timing and morphological characteristics of normal nerve growth factor-induced outgrowth, indicating that the blockade was not because of non-specific toxic effects. These results indicate that microtubule-associated protein 1B is an essential component of the molecular mechanism underlying the formation of neuronal processes.