The current study was undertaken to evaluate the anti-proliferative effect of a novel angiotensin II type 1 (AT1) receptor antagonist, RNH-6270, on exaggerated growth of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR), in comparison with the effects of an angiotensin-converting enzyme (ACE) inhibitor. RNH-6270 and temocapril significantly inhibited basal DNA synthesis in VSMCs from SHRs in a dose-dependent manner, but not in cells from Wistar-Kyoto (WKY) rats. SHR-derived VSMC showed a hyperresponse of DNA synthesis to serum and angiotensin II compared with that of WKY rats-derived VSMC. RNH-6270 did not affect serum-stimulated DNA synthesis in VSMCs from both rat strains. RNH-6270 abolished angiotensin II-stimulated DNA synthesis in VSMC from both rat strains. RNH-6270 significantly inhibited proliferation of VSMC from both rat strains, but the ACE inhibitor temocapril did not exert such an effect. RNH-6270 decreased the specific binding of angiotensin II to VSMC in a competitive manner for angiotensin II receptors in both rat strains. RNH-6270 and temocapril significantly decreased the expression of growth factor mRNAs and proteins in VSMC from SHR, but not in cells from WKY rats. These results suggest that RNH-6270 is a potent AT1 receptor antagonist and has anti-proliferative effects on VSMCs from SHR, which was not seen with an ACE inhibitor. The growth inhibitory effect of RNH-6270 may be associated with the inhibition of growth factors via antagonism to AT1 receptors.