Failing human myocardium has been associated with decreased sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity. There remains controversy as to whether the regulation of SR Ca²⁺-ATPase activity is altered in heart failure or whether decreased SR Ca²⁺-ATPase activity is due to changes in SR Ca²⁺-ATPase or phospholamban expression. We therefore investigated whether alterations in cAMP-dependent phosphorylation of phospholamban may be responsible for the reduced SR Ca²⁺-ATPase activity in human heart failure. Protein levels of phospholamban and SR Ca²⁺-ATPase, detected by Western blot, were unchanged in failing compared with nonfailing human myocardium. There was decreased responsiveness to the direct activation of the SR Ca²⁺-ATPase activity by either cAMP (0.01–100 μmol/l) or protein kinase A (1–30 μg) in failing myocardium. Using the backphosphorylation technique, we observed a decrease of the cAMP-dependent phosphorylation level of phospholamban by 20 ± 2%. It is concluded that the impaired SR function in human end-stage heart failure may be due, in part, to a reduced cAMP-dependent phosphorylation of phospholamban.