During the thermal denaturation of rabbit muscle adenylate kinase, the extents and rates of both unfolding and aggregation are dependent on protein concentration. Under identical conditions, inactivation takes place at a lower temperature than noticeable conformational changes and aggregation as measured by fluorescence, second derivative absorption spectroscopy, far ultraviolet circular dichroism and light scattering. Kinetics of inactivation can be resolved into two phases and at the same protein concentrations, the unfolding and aggregation rates are about one order of magnitude slower than the fast phase and approximately the same as the slow phase rate of the inactivation reaction between 35 and 60 degrees C. This is in general accord with the suggestion made previously that the active site of this enzyme is situated in a region more flexible than the molecule as a whole (Tsou, C.L. (1986) Trends Biochem. Sci. 11, 427-429). The inactivated enzyme cannot be reactivated by cooling and standing at 4 degrees C but can be over 80% reactivated by cooling and first standing in 3 M guanidine hydrochloride followed by diluting out the denaturant.