Studies of the folding pathway of large proteins whose kinetics is complicated due to the formation of several intermediate states are most frequently impeded or totally impossible because of rapid folding phase occurring during instrument dead time. In this paper the obtaining of energy characteristics of one of such proteins-carbonic anhydrase B-is reported. Tryptophan fluorescence and absorption methods have been used to measure the folding and unfolding kinetics of carbonic anhydrase B at different urea concentrations. In spite of the fact that the formation of the initial intermediate state of this protein takes place during the instrument dead time, the population of this state has been estimated in a wide range of urea concentrations. The use of the population of the rapidly formed intermediate state and the effective rates of slow phases of the protein folding/unfolding permitted us to calculate free energies of all the protein states and the height of energy barriers between them. It has been shown that folding of carbonic anhydrase B can be described by a consecutive reaction scheme. The possibility to obtain energy characteristics of carbonic anhydrase would allow studying structural characteristics of both intermediate and transition states via site-directed mutations. (c) 2008 Elsevier Inc. All rights reserved.