We have studied the effect of pH (6.5, 7.5), temperature (40, 50 °C), time (2-24 h) as well as lactulose (250, 450 g/L) and enzyme (3, 6, 9 U/mL) concentration on the formation of 6′-galactosyl-lactulose (β-D-Galp(1 → 6)-β-D-Galp(1 → 4)-D-Fru) and 1-galactosyl-lactulose (β-D-Galp(1 → 4)-β-D-Frup(1 → 1)-D-Gal) formed during the transgalactosylation of lactulose with β-galactosidase from Kluyveromyces lactis. The most striking feature was the different susceptibility of both trisaccharides to the reaction conditions, being 1-galactosyl-lactulose more susceptible to degradation than 6′-galactosyl-lactulose. With the exception of pH, all the parameters studied presented an important effect on the formation of both trisaccharides. Among the reaction conditions assayed, the most favourable to obtain the highest yields of 6′-galactosyl-lactulose (10.4 g/100 g of total carbohydrates) and 1-galactosyl-lactulose (11.5 g/100 g of total carbohydrates) were 50 °C, pH 6.5, 250 g/L of lactulose, 6 U/mL of enzyme and 2 h. In addition, the reaction products galactose and fructose presented an inhibitory effect on the reaction, particularly noticeable in the case of fructose. © 2011 Springer-Verlag.