Kinetic and microcalorimetric methods were used to study the interaction of thyrotoxic (V1) and normal adult cardiac myosin (V3) with ATP. It was shown that the overall enthalpy and entropy change was larger in the interaction of ATP with the thyrotoxic than with the normal myosin. There is evidence that the observed enthalpy changes were generated by protein conformational changes connected to the sequential destabilization and restabilization of the prevalent, energetically favored conformation of the myosin molecule. The V1-ATP interaction created more entropy than the V3-ATP interaction, and also the thyrotoxic isomyosin required more activation energy than the normal protein to attain the activated state. All of these results indicated that the catalytic activity of the V1 myosin was thermodynamically less efficient than that of the V3 isoenzyme.