A beta-galactosidase preparation from Bacillus circulans consists of four isoforms called beta-gal-A, beta-gal-B, beta-gal-C, and beta-gal-D. These isoforms differ in lactose hydrolysis and galacto-oligosaccharide (GOS) synthesis at low substrate concentrations. For this reason, using a selection of the isoforms may be relevant for GOS production, which is typically done at high substrate concentrations. At initial lactose concentrations in between 0.44 % and 0.68 % (w/w), beta-gal-A showed the least oligosaccharide formation, followed by beta-gal-B and beta-gal-C; most oligosaccharides were formed by beta-gal-D. The differences in behavior were confirmed by studying the thermodynamics of lactose conversion with isothermal titration calorimetry since especially beta-gal-A showed a different profile than the other isoforms. Also during the conversion of allolactose and 4-galactosyllactose at 0.44 % and 0.61 % (w/w), respectively, beta-gal-A and beta-gal-D showed clear differences. In contrast to above findings, the selectivity of the isoforms did hardly differ at an initial lactose concentration of 30 % (w/w), except for a slightly higher production of galactose with beta-gal-A. These differences were hypothesized to be related to the different accessibility of the active sites of the isoforms for different-sized reactants. The initial GOS formation rates of the isoforms indicate that beta-gal-A and beta-gal-B are the best isoforms for GOS production at high lactose concentrations.