We have examined the effects of the natriuretic peptides on DNA synthesis in primary cultures of neonatal rat cardiac fibroblasts. Binding analysis using ¹²⁵I-labeled atrial natriuretic peptide identified a single class of high-affinity binding sites (K_d=0.03±0.01 nmol/L) in these cells. Of these sites, 80% appear to be of the natriuretic peptide C receptor subtype, with the remainder being A and B receptor subtypes. Northern blot analysis confirmed the presence of all three natriuretic peptide receptors in these cells. Atrial natriuretic peptide (10⁻⁷ mol/L) effected a modest but consistent reduction in both agonist- and stretch-stimulated [³H]thymidine incorporation (17% to 41%). Moreover, brain natriuretic peptide (10⁻⁷ mol/L), C-type natriuretic peptide (10⁻⁷ mol/L), and des-[Gln¹⁸,Ser¹⁹,Gly²⁰,Leu²¹,Gly²²]-ANF 4-23-NH₂ (10⁻⁷ to 10⁻⁶ mol/L) all proved capable of antagonizing growth factor–dependent [³H]thymidine incorporation (the inhibition ranged from 14% to 28%) and cell proliferation, suggesting that all three natriuretic peptide receptor subtypes are involved in the regulation of mitogenesis in these cultures. The inhibition by atrial natriuretic peptide was amplified by cotreatment with phosphodiesterase inhibitors. Similar reduction in [³H]thymidine incorporation was seen after treatment with 8-bromo-cGMP (10⁻⁴ to 10⁻³ mol/L) or nitroprusside (10⁻⁴ to 10⁻³ mol/L). These results suggest an important paracrine role for the natriuretic peptides in regulating fibroblast growth during cardiac hypertrophy.