Ventricular hypertrophy is characterized by augmentation of synthesis, storage, and release of atrial natriuretic peptide (ANP) from ventricular tissue, but the physiological stimulus for ANP release from ventricles is not known. We determined the effect of graded, passive myocardial stretch on ANP release in isolated, arrested, perfused heart preparations after removal of the atria in 13–20-month-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). By this age, ANP gene expression was increased in the hypertrophic ventricular cells of SHR, as reflected by elevated levels of immunoreactive ANP and ANP mRNA and the increased ANP secretion (SHR, 93±14 pg/ml,n=22; WKY rats, 22±2 pg/ml,n=20;p<0.001) from perfused ventricles after removal of the atria. The release of ANP from ventricles was examined at two levels of left ventricular pressure by increasing the volume of the intraventricular balloon for 10 minutes. Stretching of the ventricles produced a rapid but transient increase in ANP secretion. As left ventricular pressure rose from 0 to 14 and 26 mm Hg in WKY rats and from 0 to 13 and 27 mm Hg in SHR, increases in ANP release into the perfusate of 1.4±0.1-fold and 1.5±0.2-fold (p<0.05) in WKY rats and 1.1±0.1-fold and 1.6±0.2-fold (p<0.05) in SHR, respectively, were observed. There was a highly significant correlation between the left ventricular pressure level and the maximal concentration of ANP in the perfusate during stretching (p<0.001,r=0.59,n=42), as well as between the maximal ANP concentrations in perfusate during stretching and the ventricular weight/body weight ratios of the corresponding animals (r=0.38,p<0.05,n=42). High performance liquid chromatographic analysis revealed that the ventricles both before and during stretch primarily released the processed, active, 28-amino acid ANP-like peptide into the perfusate. These results indicate that stretching is a direct stimulus for ventricular ANP release and show that ANP is also a ventricular hormone.