Skeletal muscle sarcoplasmic reticulum of large mammals such as rabbit contains sarcolipin (SLN), a small peptide with a single transmembrane alpha-helix. When reconstituted with the Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum into sealed vesicles, the presence of SLN leads to a reduced level of accumulation of Ca(2+). Heats of reaction of the reconstituted Ca(2+)-ATPase with ATP were measured using isothermal calorimetry. The heat released increased linearly with time over 30 min and increased with increasing SLN content. Rates ATP hydrolysis by the reconstituted Ca(2+)-ATPase were constant over a 30-min time period and were the same when measured in the presence or absence of an ATP-regenerating system. The calculated values of heat released per mol of ATP hydrolyzed increased with increasing SLN content and fitted to a simple binding equation with a dissociation constant for the SLN.ATPase complex of 6.9 x 10(-4) +/- 2.9 x 10(-4) in units of mol fraction per monolayer. It is suggested that the interaction between Ca(2+)-ATPase and SLN in the sarcoplasmic reticulum could be important in thermogenesis by the sarcoplasmic reticulum.