We have investigated the possibility that the synthesis/accumulation of neurosteroids, i.e., brain-produced steroids putatively endowed with modulatory actions in the CNS, is regulated by monoaminergic receptor-mediated mechanisms. In minces of rat brain cortex, L-ascorbic acid concentration-dependently (0.07-1.0 mM) increases the levels of pregnenolone, allotetrahydrodeoxycorticosterone, and dehydroepiandrosterone. This effect of L-ascorbic acid is region-dependent: in hippocampus, progesterone and allopregnanolone are also increased, whereas dehydroepiandrosterone is unchanged, and in corpus striatum only progesterone is increased significantly. 5-Hydroxytryptamine (10 microM), 1-(3-chlorophenyl)piperazine (1.0 microM), and 5-methoxytryptamine (0.4 microM) mimic the effect of L-ascorbic acid, whereas a pretreatment with p-chlorophenylalanine (400 mg/kg i.p., 2 days) reduces the amplitude of the L-ascorbic acid effect on brain cortical neurosteroids. The effect of L-ascorbic acid is blocked by the nonselective serotonin antagonists methiothepin, clozapine, methysergide, and pizotifen, but not mesulergine, spiperone, MDL 72222, and DL-propranolol, nor by the catecholaminergic receptor antagonists prazosin and S(-)-sulpiride. L-Ascorbic acid is not additive with dibutyryl-cyclic AMP and, furthermore, the inhibition of adenylate cyclase by MDL 12330A, but not of phospholipase C by U-73122, markedly attenuates the L-ascorbic acid-induced increase of pregnenolone in rat brain cortical minces. Together these data suggest that L-ascorbic acid plays a role in the modulation of neurosteroidogenesis, presumably by favoring the activation of the purported serotonin type 6 receptor by endogenous serotonin.