The mechanism involved in protective effect of ATP on thermal inactivation of myosin Ca-ATPase was re-examined. 1) ATP concentration required for full protection against thermal denaturation is myosin concentration dependent, and two concentrations were similar to each other. 2) The protection by ATP is more effective in the presence of Mg²⁺ than in the presence of Ca²⁺ or EDTA, suggesting the high affinity of ATP-Mg to the ATPase active site. 3) The protective effects of triphosphate compounds (ATP and AMPPNP) were distinguish-able from those of diphosphate compounds (ADP, PPi) in their concentration dependence and in the maximal protection achieved, and monophosphate compounds (AMP and IMP) were unable to protect myosin from inactivation at all.