Abnormal growth of cardiac fibroblasts is critically involved in the pathophysiology of cardiac hypertrophy/remodeling. Hexarelin is a synthetic growth hormone secretagogue (GHS), which possesses a variety of cardiovascular protective activities mediated via the GHS receptor (GHSR), including improving cardiac dysfunction and remodeling. The cellular and molecular mechanisms underlying the effect of GHS on cardiac fibrosis are however not clear. In this report, cultured cardiac fibroblasts from 8-day-old rats were stimulated with Ang II or FCS to induce proliferation. The fibroblast proliferation and DNA and collagen synthesis were evaluated utilizing MTT assay, ³H-thymidine incorporation and ³H-proline incorporation. Level of mRNA of TGF- was evaluated by RT-PCR, and active TGF-1 release from cardiac fibroblasts was evaluated by ELISA. Level of cellular cAMP was measured by radioimmunassay. In addition, the effects of DMPX (a specific adenosine receptor A₂R antagonist) and DPCPX (a specific A₁R antagonist) were tested. It was found that incubation with 10^-7 mol/L hexarelin for 24 hours 1) inhibited the Ang II-induced proliferation and collagen synthesis, and the 5% FCS and TGF-- induced increase of DNA synthesis in cardiac fibroblast; and 2) reduced Ang II-induced upregulation of TGF- mRNA expression and active TGF-₁ release from fibroblasts. Hexarelin increased the cellular level of cAMP in cardiac fibroblasts. DMPX (10^-8 mol/L), but not DPCPX, abolished the effect of hexarelin on cardiac fibroblast DNA synthesis. It is concluded that hexarelin inhibits DNA and collagen synthesis and proliferation of cardiac fibroblasts through activation of both GHSR and A₂R and diminishment of Ang II-induced increase in TGF- expression and release.