The influence of reaction conditions for oligosaccharide synthesis from stachyose using a commercial enzymatic preparation from Aspergillus aculeatus (Pectinex Ultra SP-L) was studied. Oligosaccharides were analyzed by gas chromatography with flame ionization detection (GC-FID) and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Galactosyl-melibiose (DP 3) was synthesized as a result of fructosidase activity, whereas fructosyl-stachyose (DP 5) and difructosyl-stachyose (DP 6) were formed as a consequence of the fructosyltransferase activity of Pectinex Ultra SP-L. The optimal reaction conditions for the synthesis of penta-and hexasaccharides were 60 °C, pH 5.5, 600 mg/mL stachyose, and 34 U/mL enzyme. Reaction time played an important role in oligosaccharide mixture composition constituted by 20% DP 5, 0.7% DP 6, 55% stachyose, 21% galactosyl-melibiose, and 1% monosaccharides after 1 h and 16% DP 5, 4% DP 6, 27% stachyose, 44% galactosyl-melibiose, and 2% monosaccharides after 3 h. In conclusion, stachyose could be used as a substrate for the enzymatic synthesis of new oligosaccharides that may open new opportunities in the development of future prebiotics. © 2011 American Chemical Society.