During the hydrolysis of MgATP catalyzed by myosin, ATP bound to the protein undergoes a reaction such that the beta-nonbridge oxygen atoms exchange position with the beta gamma-bridge oxygen atom. The extent of this exchange was variable but averaged 45% for ATP that had been bound for 2 s at the myosin subfragment 1 active site at ionic strength 0.08 M, pH 8.0, and 22 degrees C. This result proves that ATP cleavage in the myosin active site is readily reversible. The result also suggests that the beta-phosphate of ADP that must be formed in this cleavage step is highly constrained in the protein.