The transglycosylation reaction catalyzed by alpha-D-galactosidase from the mycelial fungus Trichoderma reesei was studied using p-nitrophenyl alpha-D-galactopyranoside (PNPG). An aliphatic alcohol or the substrate itself can be an acceptor of the galactose residue in this reaction. The transglycosylation products were identified as alkyl galactosides in the case of alcohols or as galactobioside and galactotrioside in the case of PNPG. The transglycosylation rates follow a first-order equation with respect to the alcohol concentrations except for methanol. Affinities of some substrates were estimated from their Ki values in the reaction of the enzyme with PNPG. Transglycosylation of the substrate suggests a model for the enzyme active center. It is proposed that the active center includes two galactose-binding sites and a hydrophobic site.