In the hypothalamus of the rat, the precursor of atria1 natriuretic peptide (ANP) is produced and processed into its smaller congeners of 3K mol wt species, which are secreted from neurons with cell bodies in the periventricular areas and the paraventricular nuclei of the tissue. Employing long term monolayer cultures of neonatal rat hypothalamic cells, we have identified a small population of cells that stained positive for immunoreactive (ir) ANP. Seventy-two f 7% (mean f SE; n = 4 per 1000 cells) of the irANP positive cells were colocalized with the staining of neuron-specific enolase; some of the cells possessed multiple neurites and showed irANP staining in the perikarya, in the varicosities along neuronal processes, and at the terminals of long neurites. Over the range of 10-6-10-4 M, forskolin, 3-isobutyl-l-methylxanthine, or 8-bromo-CAMP significantly augmented the total number of irANPpositive cells and those possessing neurites in a dose-related and timedependent manner. At 10-4 M, 4 days of forskolin treatment increased the number of irANP-positive neurons 4-fold (P < 0.01) while tripling that of the cells with long neurites (P < 0.01). Furthermore, it approximately tripled the number of cells (P < 0.01) showing positive signals for pro-ANP mRNA, as ascertained by calorimetric in situ hybridization using a 30.basepair antisense oligonucleotide probe labeled with digoxigenin. Consistent with the above observation, forskolin, 3-isobutyl-l-methylxanthine, or 8-bromo-CAMP treatment significantly augmented the total amount of irANP present in the cultures, with an ED50 of forskolin approximating 5 X 10-5 M. Although treatment with 10e7 M phorbol Wmyristate 13-acetate approximately doubled the production of irANP in the cultures (P < 0.05), phorbol 12-myristate 13-acetate had little effect on modulating the number or neurite outgrowth of irANP neurons. Thus, our present findings suggest that protein kinase-A pathways are of greater importance than protein kinase-C pathways in regulating both the functional and morphological development of ANP-producing neurons during the ontogenesis of the rat hypothalamus.