A study on optimisation of the conditions for galactooligosaccharide (GOS) formation during lactose hydrolysis, produced by Lactozym 3000 L HP G, was carried out. The synthesis was performed during times up to 300 min at 40, 50 and 60 °C, pH 5.5, 6.5 and 7.5, lactose concentration 150, 250 and 350 mg/mL and enzyme concentration 3, 6 and 9 U/mL. The product mixtures were analysed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). During the hydrolysis of lactose, besides glucose and galactose, galactobiose, allolactose and 6′ galactosyl lactose were also formed as a result of transgalactosylation catalysed by the enzyme. The effect of the reaction conditions was different in the formation of di- and the trisaccharide. Thus, the optimal conditions for galactobiose and allolactose synthesis were 50 °C, pH 6.5, 250 mg/mL of lactose, 3 U/mL of enzyme and 300 min, whereas the best reaction conditions for 6′ galactosyl lactose production were 40 °C, pH 7.5, 250 mg/mL of lactose, 3 U/mL of enzyme and 120 min. These results show the possibility to obtain reaction mixtures with Lactozym 3000 L HP G, with different composition, depending on the assayed conditions. © 2007 Elsevier Ltd. All rights reserved.