There are two kinds of conformational forms of adenylate kinase (AK) in equilibrium in solution with different ANS-binding properties. Furthermore, the nature of AP 5 A inhibition suggests also that the native forms of AK for binding with different substrates pre-exist in the absence of substrates. In the present study, a kinetics approach was used to explore the native forms distinguished by ANS-binding properties and by the nature of AP 5 A inhibition. The results revealed that the native forms distinguished by ANS probe are two conformational sub-ensembles. Both sub-ensembles are active and consist of a series of forms, which pre-exist in solution and can bind with different substrates. The K m values of N 1 for AMP, ADP and MgATP are larger than that of N 2 , indicating that the N 2 sub-ensemble is more specific for binding substrates. This is consistent with the previous observation that the activity of N 2 is about 1.8-fold of that of N 1 .