Phospholamban is the regulator of Ca²⁺-ATPase in cardiac sarcoplasmic reticulum(SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban. Phosphorylation of phospholamban relieves this inhibition. Recently, there has been a report that the cytoplasmic domain (amino acids 1-25) of phospholamban is insufficient to inhibit the Ca²⁺ pump. To explore the domains of phospholamban responsible for Ca²⁺-ATPase inhibitory activity, we examined the effect of a synthetic phospholamban peptide consisting of amino acid residues 1-25 on Ca²⁺ uptake by reconstituted skeletal SR Ca²⁺-ATPase. The Ca²⁺-ATPase of skeletal SR was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC) or phosphatidylcholine: phosphatidylserine (PC:PS). Inclusion of a phospholamban peptide in PC proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺ uptake at pCa 6.0, and phosphorylation of this peptide by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effect on the Ca²⁺ pump. Similar effects of phospholamban peptide were also observed using PC:PS proteoliposomes. Based on these results, we could conclude that the cytoplasmic domain of phospholamban, containing the phosphorylation sites, by itself is sufficient to inhibit the Ca²⁺ pump of SR.