The activity loss of creatine kinase (CK), observed at low pH (midpoint was 4.8) corresponded to the monomerization of the dimeric protein and was correlated with structural changes. The acid-induced unfolding was not complete at this pH, as probed by circular dichroic (CD) and fluorescence methods. Further decrease of pH, led to a second transition (midpoint was pH 3.5). The loss of activity was irreversible at pH 4.8 (< 20% native activity was recovered) while it was almost fully reversible (> 90% of native activity was recovered) for the enzyme incubated at pH 0.9-2.5. The amount of intermolecular beta-sheets (monitored with the 1620 cm-1 infrared component band) was maximal when the enzyme was incubated at pH 4.8, as a consequence of protein aggregation, while it was minimal at extremes of pH and at low ionic strength. Acid-induced and alkaline-induced denaturations promoted different structural changes, leading to distinct partially unfolded conformational states. The addition of KCl (from 0.05 M to 0.5 M) to an acidic solution of monomeric creatine kinase (pH 1.6) resulted in a highly cooperative transition from the partially unfolded conformation (UA) to the more compact conformation (A) with the properties of a molten globule, as probed by CD spectra and by fluorescence. The formation of intermolecular beta-sheets in the compact conformation was observed by infrared spectroscopy, indicating formation of unstable aggregates.