Besides acting as an important cofactor in the biosynthesis of catecholamine, ascorbic acid (AA) also modulates the activity of peptidyl- glycine α-amidating monooxygenase for the post-translational modification of neuropeptides such as α-MSH and TRH. We report here a novel action of AA in modulating the secretion and mRNA expression of atrial natriuretic factor (ANF) in rat hypothalamic neurons. Primary cultures of hypothalamic neurons from neonatal rats as previously described were employed in the present studies. Six days after plating, cultures were replenished with serum free media and incubated with vehicle or various doses of AA, alone or in the presence of forskolin. Treatment with AA alone significantly increased irANF secretion from the cultures in a time-related and a dose-dependent manner with an ED50 of approximately 3 μM and an Emax of 100 μM. At the concentration of 10 μM, AA augmented irANF release approximately 3 fold that of the controls (55 ± 7 pg/well; mean ± SE, n = 3; P<0.01), but it failed to affect the abundance of pro-ANF mRNA in the cultures. However, 10 μM of AA markedly enhanced forskolin-induced irANF secretion and pro-ANF mRNA abundance of the cultured cells. This potentiating effect of AA on forskolin stimulation showed a good parallelism to the levels of cAMP produced in the hypothalamic cultures. We thus conclude that AA acts alone or in synergism with forskolin to stimulate the secretion and production of ANF in rat hypothalamic neurons; this latter effect may operate at the genomic level and is mediated, at least in part, through the protein kinase A dependent pathway.