This report measured the amount of heat released during Ca(2+) transport and ATP hydrolysis by vesicles derived from the sarcoplasmic reticulum of rabbit slow (SM) and fast (FM) muscle. During ATP hydrolysis, part of the chemical energy released is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles), the heat produced per mol of ATP cleaved by SM and FM vesicles was the same and varied between 7.7-9.1 kcal. In the presence of the gradient the heat produced by SM and FM vesicles differed, 13.4 kcal/mol and 23.0 kcal/mol ATP cleaved, respectively. After formation of the gradient, part of the ATPase activity was not coupled to Ca(2+) transport. The difference of heat produced by FM and SM vesicles during ATP hydrolysis was related to the rate of uncoupled ATPase activity. When extended to the living cell, the data described indicate that the amount of heat produced by the Ca(2+)-ATPase of SM muscle is 36 times smaller than that produced by the FM. Thyroid hormone 3,5,3'-triiodo L-thyronine (T3) regulate both thermogenesis and the transcription of the sarcoplasmic reticulum Ca(2+)-ATPase isoforms found in SM and FM. The findings described in this report raise the possibility that one of the mechanisms of thermogenesis control may be related to the regulation of Ca(2+)-ATPase isoforms expression by T3.