The aim of this research is to quantify the effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Quantification of these effects is important because temperature and enzyme origin are important process parameters, A kinetic model was used to describe the concentrations in time. The kinetic parameters were determined by using data obtained in batch experiments at various temperatures (20, 30, 40, and 50 degrees C) and by using beta-galactosidases from Bacillus circulans, Aspergillus oryzae, Kluyveromyces lactis, and Kluyveromyces fragilis. The effect of temperature on the kinetic parameters could be described with the Arrhenius equation, except for the inhibition parameter. Slightly higher oligosaccharide yields were found at higher temperatures. However, the influence of the initial lactose concentration was much larger. The higher yield at higher temperatures is an additional advantage when operating at high initial lactose concentrations and consequently elevated temperatures. Clear differences between the beta-galactosidases were found concerning amount, size, and type of oligosaccharides produced. The beta-galactosidase from B. circulans produced the most abundant amount, the most different, and largest-sized oligosaccharides. The beta-galactosidases from Kluyveromyces spp. produced mainly trisaccharides. The kinetic parameters for the different enzymes were determined and differences were discussed.