AbstractPrevious studies have shown that atrial natriuretic peptides inhibit mitogenesis in subcultured aortic smooth muscle cells by a mechanism that appears to be mediated via the C‐type or “clearance” receptor. In the current study, we have compared the antimitogenic effect of these peptides in serum‐stimulated primary aortic smooth muscle cell cultures and in subcultured cells. A series of atrial peptides, including rANF99–126, rANF103–126, and rANF103–125, were only poorly antimitogenic in serum‐stimulated primary cultures, whereas des[Cys105, Cys121] rANF104–126which binds selectively to the ANF‐C receptors had no antimitogenic activity. In contrast, in subcultured cells (between subcultures 3 and 25), rANF99–126, rANF103–126, rANF103–126, Cys116rANF102–116, and des[Cys105, Cys121]rANF104–126inhibited serum‐induced [3H]thymidine incorporation (IC50in the range of 10–50 nM), with maximal inhibition of 40–70%. The lack of antimitogenic activity in primary cultures did not appear to be related to the lack of cGMP elevation elicited by atrial peptides or to an inherent insensitivity to the action of antimitogens, because primary cultures were responsive to the cGMP‐elevating effect of atrial peptides and the cells were more rather than less sensitive to the antimitogenic effect of the nitric‐oxide‐vasodilator, SNAP, as compared to subcultured cells. Analysis of the affinity and binding capacity of freshly isolated aortic membranes, and primary or secondary cultures for [125I]rANF99–126, revealed that the number of ANF receptors increased by tenfold, following subculture. Moreover, subcultured cells contained receptors with increased binding affinity for peptide analogues selective for the ANF‐C‐type type receptor. Covalent cross‐linking studies with (125I)rANF99–126confirmed that membranes prepared from fresh aortae predominantly expressed the ANF‐A/guanylate cyclase receptor, whereas in subcultured cells the predominantly cross‐linked protein was the ANF‐C‐type receptor, with receptors in primary cultures occupying an intermediate position. These results suggest that the binding and antimitogenic activity of atrial peptides in aortic smooth muscle cells depends on the phenotypic state of these cells. Moreover, the increased antimitogenic potency of atrial peptides in secondary cultures may reflect increased exp