The current studies were designed to explore the effects of C-receptor-binding atrial natriuretic peptide analogues on serum-induced mitogenesis in cultured rat aortic smooth muscle cells. To this end, rANF_99–126 and a series of truncated (rANF_103–125, rANF_103–125), ring-deleted (des[Gln¹¹⁶, Ser¹¹⁷, Gly¹¹⁸, Leu¹¹⁹, Gly¹²⁰]rANF_102–121-NH₂ (c-ANF) and linear des(Cys¹⁰⁵, Cys¹²¹)rANF_104–126 peptide analogues were used. The latter two peptides have been reported to be selective for the ANF-C receptor. In cells subcultured between passage 3 to 19, rANF_99–126, rANF_103–126, and rANF_103–125 concentration-dependently (0.1–1000 nM) inhibited serum-induced (³H) thymidine incorporation with maximal inhibition observed at 1 μM for each peptide (∼40, 31 and 56%) respectively. Furthermore, des[Cys¹⁰⁵, Cys¹²¹]rANF_104–126 inhibited serum-induced (³Hthymidine incorporation concentration-dependently without altering basal or elevated cellular cAMP or cGMP levels. Moreover, the reduction in thymidine incorporation was associated with inhibition of serum-induced clonal cell proliferation. In contrast, c-ANF failed to inhibit serum-induced mitogenesis, yet at a concentration of 100 nM it antagonized the antimitogenic effects of des[Cys¹⁰⁵, Cys¹²¹]rANF_104–126 or rANF_99–126 without having any effect on basal or elevated cellular cyclic nucleotide levels. We conclude that the antimitogenic effect of atrial peptides is mediated through interaction with the ANF-C receptor and may be independent of changes in cellular cyclic nucleotide levels.