We have shown that the tetrapeptide Ac-SDKP inhibited endothelin 1 (ET-1)-induced cell proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFs) and reduced left ventricle collagen deposition in rats with aldosterone-salt- and angiotensin II (Ang II)-induced hypertension. However, it is not known whether these effects are mediated by receptor binding sites specific for Ac-SDKP. We hypothesized that Ac-SDKP exerts anti-fibrotic effects by binding to specific receptor sites in cultured rat CFs, which mediate the inhibitory effects of Ac-SDKP on ET-1-stimulated collagen synthesis. Ac-SDKP binding sites in rat CFs and hearts were characterized using a specific radioligand, [¹²⁵I]-Hpp-Aca-SDKP, a biologically active analogue of Ac-SDKP. [¹²⁵I]-Hpp-Aca-SDKP bound to rat CFs and fractionated membranes with similar affinities and specificity and in a concentration- and time-dependent fashion. Scatchard plot analysis revealed a single class of high-affinity Hpp-Aca-SDKP binding sites (B_max: 1704 ± 198 fmol/mg protein; K_d: 3.3 ± 0.6 nM). [¹²⁵I]-Hpp-Aca-SDKP binding in CFs was displaced by unlabeled native peptide Ac-SDKP (K_i: 0.69 ± 0.15 nM) and the analogue Hpp-Aca-SDKP (K_i: 10.4 ± 0.2 nM), but not the unrelated peptide Ang II or ET-1 (10 µM). In vitro, both Ac-SDKP and Hpp-Aca-SDKP inhibited ET-1-stimulated collagen synthesis in CFs in a dose-dependent fashion, reaching a maximal effect at 1 nM (control: 7.5 ± 0.4; ET-1: 19.9 ± 1.2; ET-1±SDKP: 7.7 ± 0.4; ET-1+Hpp-Aca-SDKP: 9.7 ± 0.1 mg/mg protein; p <0.001). Ac-SDKP also significantly attenuated ET-1-induced increases in intracellular calcium and mitogen-activated protein kinase ERK 1/2 phosphorylation in CFs. In the rat heart, in vitro autoradiography revealed specific [¹²⁵I]-Hpp-Aca-SDKP binding throughout the myocardium, primarily interstitial in location. We believe these results demonstrate for the first time that Hpp-Aca-SDKP is a functional ligand specific for Ac-SDKP receptor binding sites, and that both Ac-SDKP and Hpp-Aca-SDKP exert anti-fibrotic effects by binding to Ac-SDKP receptors in rat CFs.