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The myofibril is a good model to study the ATPase of the muscle fibre. When myofibrillar ATPase reaction mixtures are quenched in acid, there is a burst of P_i formation, due to AM · ADP · P_i or P_i as shown in the scheme: AM + ATP ↔ A·M·ATP ↔ AM·ADP·Pi ↔ AM·ADP + P_i ↔ AM + ADP. Therefore, in the steady state, either AM·ADP·P_i or AM·ADP or both predominate. To determine which, we studied the reaction using a P_i binding protein (from E. coli) labeled with a fluorophore such that it is specific and sensitive to free P_i [Brune, M. et al. (1994) Biochemistry 33, 8262–8271]. We show that the P_i bursts with myofibrillar ATPases (calcium‐activated or not, or crosslinked) are due entirely to protein bound P_i. Thus, with myofibrillar ATPases the AM·ADP·P_i state predominates.