Many b-galactosidases show large differences in galacto-oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various b-galactosidases is quantified. The use of oNPG as a competing substrate to lactose yields more information than can be obtained by examining only the conversion of lactose itself. The reaction rate with lactose or oligosaccharides as substrate relative to that with water as acceptor is much higher for the b-galactosidase of Bacillus circulans than the bgalactosidases of Aspergillus oryzae and Kluyveromyces lactis. In addition, the bgalactosidase of B.circulans has a high reaction rate with galactose as acceptor, in contrast to those of A. oryzae and K. lactis. The latter two are strongly inhibited by galactose. These differences explain why b-galactosidase of B. circulans gives higher yields in GOS production than other b-galactosidases. Many of the reaction rate constants for the b-galactosidase isoforms of B. circulans increase with increasing molecular weight of the isoform. This indicates that the largest isoform b-gal-A is most active in GOS production. However, its hydrolysis rate is also much higher than that of the other isoforms, which results in a faster hydrolysis of oligosaccharides as well.