The β-galactosidase activity of the commercial enzymatic preparation Pectinex Ultra SP-L derived from Aspergillus aculeatus has been used to hydrolyze and transgalactosylate the prebiotic carbohydrate lactulose. During this reaction, new oligosaccharides derived from lactulose have been detected by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The presence of the trisaccharide 6′-galactosyl- lactulose, the major compound formed, has been confirmed by NMR. In addition, disaccharides and other oligosaccharides with higher retention times have been also detected. The effect of transgalactosylation conditions such as time, temperature, pH, and initial lactulose and enzyme concentrations, as well as product inhibition on oligosaccharide synthesis, has been studied. The optimal conditions for the formation of tri and higher oligosaccharides were 60 °C, pH 6.5, 450 g/L lactulose, 16 units/mL of enzyme, and 7 h of reaction. Selective formation of disaccharides was achieved under the same conditions with the exception of pH (4.5). The present work provides additional knowledge on the synthesis of new oligosaccharides with potential prebiotic properties. © 2008 American Chemical Society.