Enzymatic properties of the a-galactosidase (a-galactoside galactchydrolase, EC 3.2.1.22) from Trichoderma reesei in the hydrolysis of natural galactooligosaccharides and alpha-O-methyl D-galactopyranoside have been investigated in a wide range of substrate concentrations. The hydrolyses of alpha-O-methyl D-galactopyranoside and melibiose were inhibited by substrate at concentrations higher than 100 mM while in the hydrolysis of raffinose and stachyose such an effect was not observed. It was shown by H-1 and C-13 NMR spectroscopy and HPLC techniques that inhibition by the excess of alpha-O-methyl D-galactopyranoside or melibiose strongly correlated with formation of transglycosylation products. The product of autocondensation reaction with alpha-O-methyl D-galactopyranoside as substrate was found to be alpha-O-methyl galactopyranosyl-1,6-D-galactopyranoside. The stereochemical course of stachyose hydrolysis has been determined. The enzyme catalyses the hydrolysis with retention of anomeric configuration and is assumed to operate via a double displacement mechanism. Simultaneous hydrolysis of stachyose and raffinose effected by the a-D-galactosidase was studied by direct H-1 NMR measurements. Cleavage of the terminal galactose residue of stachyose was found to be the rate-limiting step. Formation constants of enzyme-substrate complex for stachyose and raffinose were calculated. The suggested model can be used for simulating the two-substrate system and predicting the extent of stachyose hydrolysis.