It is commonly believed that MgATP2− is the substrate of F1‐ATPases and ATP4− acts as a competitive inhibitor. However, the velocity equation for such competitive inhibition is equivalent to that for a rapid equilibrium ordered binding mechanism in which ATP4− adds first and the binding of Mg2+ is dependent on the formation of the E ATP4− complex. According to this ordered‐binding model, solution formed MgATP2− is not recognized by the ATPase as a direct substrate, and the high‐affinity binding of Mg2+ to the E ATP4− complex is the key reaction towards the formation of the ternary complex. These models (and others) were tested with an F1‐ ATPase, isolated from Halobacterium saccharovorum, by evaluating the rate of ATP hydrolysis as a function of free ATP4− or free Mg2+. The rates were asymmetrical with respect to increasing ATP4− versus increasing Mg2+. For the ordered‐binding alternative, a series of apparent dissociation constants were obtained for ATP4− (.cf2.K.cf2..cf1..esapp.rb.eiA.rb), which decreased as Mg2+ increased. From this family of .cf2.K.cf2..cf1..esapp.rb.eiA.rb the true KA was retrieved by extrapolation to Mg2+ = 0 and was found to be 0.2 mM. The dissociation constants for Mg2+, established from these experiments, were also apparent (.cf2.K.cf2..cf1..esapp.rb.eiB.rb) and dependent on ATP4− as well as on the pH. The actual KB was established from a series of .cf2.K.cf2..cf1..esapp.rb.eiB.rb by extrapolating to ATP4− = ∞ and to the absence of competing protons, and was found to be 0.0041 mM. The pKa of the protonable group for Mg2+ binding is 8.2. For the competitive inhibition alternative, rearrangement of the constants and fitting to the velocity equation gave an actual binding constant for MgATP2− (KEAB) of 0.0016 mM and for ATP4− (KEA) of 0.2 mM. Decision between the two models has far‐reaching mechanistic implications. In the competitive inhibition model MgATP2− binds with high affinity, but Mg2+ cannot bind once the E ATP4− complex is formed, while in the ordered‐binding model binding of Mg2+ requires that ATP4− adds first. The steric constraints evident in the diffraction structure of the ATP binding site in the bovine mitochondrial F‐ATPase Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621−628 tend to favor the ordered‐binding model, but the final decision as to which kinetic model is valid has to be from further structural studies. If the ordered‐binding model gains more experimental support, a revision of the current concepts of unisite catalysis and negative cooperativity of nucleotide binding will be necessary.