Both sarcolipin (SLN) and phospholamban (PLN) lower the apparent affinity of either SERCA1a or SERCA2a for Ca(2+). Since SLN and PLN are coexpressed in the heart, interactions among these three proteins were investigated. When SERCA1a or SERCA2a were coexpressed in HEK-293 cells with both SLN and PLN, superinhibition resulted. The ability of SLN to elevate the content of PLN monomers accounts, at least in part, for the superinhibitory effects of SLN in the presence of PLN. To evaluate the role of SLN in skeletal muscle, SLN cDNA was injected directly into rat soleus muscle and force characteristics were analyzed. Overexpression of SLN resulted in significant reductions in both twitch and tetanic peak force amplitude and maximal rates of contraction and relaxation and increased fatigability with repeated electrical stimulation. Ca(2+) uptake in muscle homogenates was impaired, suggesting that overexpression of SLN may reduce the sarcoplasmic reticulum Ca(2+) store. SLN and PLN appear to bind to the same regulatory site in SERCA. However, in a ternary complex, PLN occupies the regulatory site and SLN binds to the exposed side of PLN and to SERCA.