The neurosteroid pregnenolone (PREG) and its chemically synthesized analog 3beta-methoxypregnenolone (MePREG) bind to microtubule-associated protein 2 (MAP2) and stimulate the polymerization of microtubules. PREG, MePREG, and progesterone (PROG; the physiological immediate metabolite of PREG) significantly enhance neurite outgrowth of nerve growth factor-pretreated PC12 cells. However, PROG, although it binds to MAP2, does not increase the immunostaining of MAP2, contrary to PREG and MePREG. Nocodazole, a microtubule-disrupting agent, induces a major retraction of neurites in control cultures, but pretreatment with PREG/MePREG is protective. Decreasing MAP2 expression by RNA interference does not modify PROG action, but it prevents the stimulatory effects of PREG and MePREG on neurite extension, showing that MAP2 is their specific receptor.