The present studies were undertaken to investigate the effect of C-ANP4-23, and several peptide fragments containing 12 amino acids from different regions of cytoplasmic domain of NPR-C on cell proliferation in the absence or presence of angiotensin II (Ang II), endothelin (ET-1) and arginine-vasopressin (AVP) in A10 vascular smooth muscle cells (VSMC). The peptide fragments used have either complete Gi activator sequences K⁴⁶¹-H⁴⁷² (peptide-1) and H⁴⁸¹-H⁴⁹³ (peptide 3) or partial Gi activator sequences R⁴⁶⁹-K⁴⁸⁰ (peptide 2) and I⁴⁶⁵-H⁴⁷² (peptide Y) with truncated carboxyl or amino terminus respectively. The other peptide used had no structural specificity Q⁴⁷³-K⁴⁸⁰ (peptide X) or was the scrambled peptide control for peptide-1 (peptide Z). Ang II, ET-1 and AVP significantly stimulated DNA synthesis in these cells as determined by ³H-thymidine incorporation that was inhibited by peptides 1, 2 and 3 and not by peptides X, Y and Z in a concentration-dependent manner, with an apparent Ki between 1-10 nM. In addition, C-ANP_4-23, that interacts with NPR-C, also inhibited DNA synthesis stimulated by vasoactive peptides; however, the inhibition elicited by C-ANP_4-23 was not additive with the inhibition elicited by peptide-1. On the other hand, basal DNA synthesis in these cells was not inhibited by C-ANP4-23 or by the peptide fragments. Furthermore, vasoactive peptide-induced stimulation of DNA synthesis was inhibited by PD98059 and wortmannin, and this inhibition was potentiated by peptide-1. In addition, peptide-1 also inhibited vasoactive peptide-induced phosphorylation of ERK1/2 and AKT and enhanced expression of Gi proteins. These data suggest that C-ANP_4-23 and small peptide fragments containing 12 amino acids irrespective of the region of cytoplasmic domain of NPR-C inhibit the proliferative responses of vasoactive peptides through Gi protein and MAP kinase/PI3K/AKT pathways.