The molecular mechanism of movement has been studied widely. The energy needed for movement comes from ATP hydrolysis mediate by myosin, a primary energy transducer in muscle. There are several enzymatic systems that can supply ATP. The synthesis by these systems occurs with heat absorption. The aim of this work was to determine whether myosin could capture, cleave ATP and generate heat from two different ATP regenerator systems: hexokinase (HK) and pyruvate kinase (PK).  In the absence of glucose, HK generates ATP from glucose 6-phosphate (G6P) and ADP. PK produces ATP from phosphoenolpyruvate (PEP) and ADP during glycolysis. We performed experiments using skeletal muscle myosin subfragment–1 (S1) which contains the actin-binding site and the catalytic site.  ATPase activity was measured by Pi release (Fiske and Subbarow, 1925) and heat generation was quantified using an OMEGA Isothermal Titration Calorimeter from Microcal Inc., at pH 7.0 and 35 ^oC. The calorimetric enthalpy (∆H^cal) was calculated by dividing the amount of heat released by the amount ATP hydrolyzed. In the PK system, ATP cleavage (0.14± 0.02 mmol Pi/mg.min) was higher than with the HK system (0.06 ±0.01 mmol Pi/mg.min) and the latter activity was inhibited by glucose. The energetic balance between ATP production by PK and ATP consumption by S1 was more exothermic (∆H^cal = -4.69±0.36 kcal/mol) than HK (∆H^cal = +0.27±0.30 kcal/mol). This difference can be explained because PEP is a higher-energy compound than G6P. With ample exogenous ATP (no HK or PK), ATPase activity was 0,14 ±0,02 mmol Pi/mg.min, similar to that seen with PK system. However, heat production was two-fold greater with exogenous ATP. Our results suggest that the HK and PK systems produce enough ATP to sustain hydrolysis by S1. Thus under certain conditions the organism might be able to use them as an energy source.