Endo beta-1.3-1.4-glucanase Lic16A of the moderate thermophilic anaerobe Clostridium thermocellum has a complex multimodular structure. In addition to the catalytic module it contains 8 auxiliary modules, 5 of which are substrate binding modules. The new family 54 substrate binding module CBM54 (25.2 kDa), localized at the N-terminus of the enzyme, owns a cleavage site, in its N-terminal part manifested by the appearance of a shortened module CBM54C (17.2 kDa) in vivo and in vito. CBM54C was cloned in Escherichia coli cells and purified to electrophoretic homogeneity. The binding constants of CBM54C to xylan, chitin, insoluble B-glucan of yeast cell wall and bacterial crystalline cellulose were in the same order of magnitude as for CBM54. However CBM54C, unlike CBM54, did not bind pustulan, avicel, and chitosan. Nevertheless the presence of calcium ions restored the ability of CBM54C to bind the latter three carbohydrates. CBM54 substrate binding promiscuity permits to suggest the presence of multiple binding sites, some of them calcium dependent. The collected data allow to localize Ca2+ -independent sites for avicel, pustulan an d chitosan binding inthe spontaneously split-off CBM54 N-terminal area (8 kDa). In this report the localization scheme of Ca2+ -dependent and Ca2+ -independent binding sites for various substrates has been suggested.