The presence of calcium stimulated adenosine triphosphatase (Ca2+,Mg(2+)-ATPase) activity in isolated transverse tubule (t-tubule) membranes is distinguished from magnesium adenosine triphosphatase (Mg(2+)-ATPase) activity on the basis of differing thermal stabilities. The Mg(2+)-ATPase is the major protein component of the t-tubule membrane, and it can be difficult to discriminate between the low levels of Ca2+ stimulated ATPase activity found in isolates of t-tubules compared to the much higher Mg(2+)-ATPase activity. Thermal analysis reveals different inactivation temperatures (Ti) for the proteins responsible for ATP dependent calcium transport (Ti = 49 degrees C) and Mg(2+)-ATPase activity (Ti = 57 degrees C) in isolated t-tubule membranes. The differential scanning calorimetry profile of t-tubule membranes consists of three major components with transition temperatures (Tm) of 51 degrees C, 57 degrees C and 63 degrees C. Denaturation of the component with Tm = 57 degrees C correlates with inactivation of Mg(2+)-ATPase activity, and denaturation of the Tm = 51 degrees C component correlates with the inactivation of Ca2+,Mg(2+)-ATPase activity and calcium transport. The functions of the t-tubule membrane component or components that denature with Tm = 63 degrees C have yet to be identified. The lack of stimulation of calcium transport in isolated t-tubules by oxalate, the impermeability of isolated t-tubules to oxalate, and experiments performed on t-tubules with defined amounts of sarcoplasmic reticulum (SR) added suggest that contamination of the isolated t-tubules by SR is unlikely to account for the level of Ca2+,Mg(2+)-ATPase activity detected. The presence of a Ca2+,Mg(2+)-ATPase in the t-tubule membrane would provide a mechanism that may be involved in the partial removal of calcium that is accumulated in the junctional space during muscle relaxation or calcium that is released from the terminal cisternae of sarcoplasmic reticulum during excitation-contraction coupling.