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Three different fungal b-galactosidases, from three commercial sources, were studied for their capacity to produce galacto-oligosaccharides by trans-galactosylation from lactose. The enzymes were investigated in a soluble form for the GOS production at two concentrations of lactose (10 % and 30 % w/w). The maximum of GOS concentration (70.9 g·L -1 ) was obtained at 30 % of the initial lactose (pH 4.5, temperature 30 °C) in the second hour of enzyme reaction. The GOS yield (24 % w/w) was not significantly affected by the process of immobilisation in comparison with the free enzyme preparation (22 % w/w), but time to obtain a maximal GOS concentration was prolonged. Galacto-oligosaccharides were effectively produced by immobilised b-galactosidase in the 15 repeated batch runs of 30 % (w/w) of lactose as the substrate.