In the present studies the chronic effects of glucocorticoids and drugs activating cAMP-dependent pathways on the production and secretion of immunoreactive (ir) ANP from long term monolayer cultures of neonatal rat hypothalamic neurons were examined. Forskolin treatment increased ir-ANP release in a time-dependent and dose-related manner, with an EC50of approximately 30 μM; at a lower dose of 10 μM, forskolin doubled ir-ANP release (P < 0.01) compared to that in control cultures (mean ± SEM, 9.6 ± 0.3 pg/well; n = 4). While dexa-methasone (DM) alone did not affect basal secretion of ir-ANP, 10 nM of the glucocorticoid significantly enhanced the effect of forskolin (10 μm) by raising ir-ANP release approximately 3 times that induced by forskolin alone (P < 0.001). This potentiation of DM was both time dependent and dose responsive, with an EC50of 1 nM; this effect was significantly suppressed by 100 nM RU38486, a glucocorticoid or type II receptor antagonist, but not by RU28318, a mineralocorticoid receptor antagonist. In addition, forskolin (10 μm) or DM (10 nM) alone significantly increased ir-ANP production approximately 1.4 times (P < 0.05) and 1.3 times (P < 0.05) over that of control cultures, respectively, whereas concurrent treatment with forskolin and DM increased ir-ANP production by approximately 1.8 times (P < 0.01). These changes were reflected by a corresponding increment in the abundance of pro-ANP mRNA in the cultures, as demonstrated by Northern blot analysis. We conclude from the present findings that glucocorticoid-and cAMP-dependent pathways may modulate the function of ANP neurons in rat hypothalami by regulating the secretion and production of the neuropeptide at the genomic level.