—Three weeks after myocardial infarction (MI) in the rat, remodeled hypertrophy of noninfarcted myocardium is at its maximum and the heart is in a compensated stage with no evidence of heart failure. Our hemodynamic measurements at this stage showed a slight but insignificant decrease of +dP/dt but a significantly higher left ventricular end-diastolic pressure. To investigate the basis of the diastolic dysfunction, we explored possible defects in the β-adrenergic receptor–G_s/i protein–adenylyl cyclase–cAMP–protein kinase A–phosphatase pathway, as well as molecular or functional alterations of sarcoplasmic reticulum Ca²⁺-ATPase and phospholamban (PLB). We found no significant difference in both mRNA and protein levels of sarcoplasmic reticulum Ca²⁺-ATPase and PLB in post-MI left ventricle compared with control. However, the basal levels of both the protein kinase A–phosphorylated site (Ser16) of PLB (p16-PLB) and the calcium/calmodulin–dependent protein kinase-phosphorylated site (Thr17) of PLB (p17-PLB) were decreased by 76% and 51% in post-MI myocytes (P<0.05), respectively. No change was found in the β-adrenoceptor density, G_sα protein level, or adenylyl cyclase activity. Inhibition of phosphodiesterase and G_i protein by Ro-20-1724 and pertussis toxin, respectively, did not correct the decreased p16-PLB or p17-PLB levels. Stimulation of β-adrenoceptor or adenylyl cyclase increased both p16-PLB and p17-PLB in post-MI myocytes to the same levels as in sham myocytes, suggesting that decreased p16-PLB and p17-PLB in post-MI myocytes is not due to a decrease in the generation of p16-PLB or p17-PLB. We found that type 1 phosphatase activity was increased by 32% (P<0.05) with no change in phosphatase 2A activity. Okadaic acid, a protein phosphatase inhibitor, significantly increased p16-PLB and p17-PLB levels in post-MI myocytes and partially corrected the prolonged relaxation of the [Ca²⁺]_i transient. In summary, prolonged relaxation of post-MI remodeled myocardium could be explained, in part, by altered basal levels of p16-PLB and p17-PLB caused by increased protein phosphatase 1 activity.