The incorporation of32P1into phospholamban, troponin I, phosphatidylinositols, and inositol trisphosphates was studied in Langendorff-perfused guinea pig hearts stimulated with isoproterenol. Hearts were perfused with Krebs-Henseleit buffer containing [32P]P|and freezeclamped at different times during the positive inotropic response. Exposure of the hearts to 0.1 μM isoproterenol for up to 1 minute was associated with significant (up to threefold) increases in phospholamban and troponin I phosphorylation, but there was no significant increase in32P incorporation into phospholipids. However, longer exposure (2 minutes or more) to isoproterenol was associated with increases in the degree of P labeling of phosphatidylinositols and phosphatidic acid. Examination of32P labeling of Inositol trisphosphates in the same hearts revealed that the radioactivity associated with these compounds decreased with time. The decreases were significant at times of exposure of 2 minutes or longer to β-adrenergic stimulation. The tissue levels of the inositol 1,4,5-trisphosphate isoform were also measured in hearts perfused with isoproterenol for 3 minutes, and they were found to be significantly lower compared with values obtained in control hearts. The effects of isoproterenol on32P incorporation into phospholipids and proteins were observed in the presence of prazosin, and they were completely abolished by the β-receptor blocker propranolol. Examination of the phosphoinositldespecific phosphoUpase C activity in the perfused hearts revealed that isoproterenol stimulation was associated with a decrease in the membrane-associated enzymatic activity at physiological calcium concentrations. These findings indicate that β-adrenergic stimulation of isolated hearts is associated with changes in basal phosphatidylinositol turnover that may be mediated, at least in part, by inhibition of the phosphoUpase C enzymatic activity specific for phosphatidylinositols.