Using a chemically defined reconstitution system, we performed a systematic study of key factors in the regulation of the Ca-ATPase by phospholamban (PLB). We varied both the lipid/protein and PLB/Ca-ATPase ratios, determined the effects of PLB phosphorylation, and compared the regulatory effects of several PLB mutants, as a function of Ca concentration. The reconstitution system allowed us to determine accurately not only the PLB effects on KCa (Ca concentration at half-maximal activity) of the Ca-ATPase, but also the effects on Vmax (maximal activity). Wild-type PLB (WT-PLB) and two gain-of-function mutants, N27A-PLB and I40A-PLB, showed not only the previously reported increase in KCa, but also an increase in Vmax. Specifically, Vmax increases linearly with the intramembrane PLB concentration, and is approximately doubled when the sample composition approaches that of cardiac SR. Upon phosphorylation of PLB at Ser-16, the KCa effects were almost completely reversed for WT- and N27A-PLB but were only partially reversed for I40A-PLB. Phosphorylation induced a Vmax increase for WT-PLB, and a Vmax decrease for N27A-and I40A-PLB. We conclude that PLB and PLB phosphorylation affect Vmax as well as Kca, and that the magnitude of both effects is sensitive to the PLB concentration in the membrane.