Production of galacto-oligosaccharide (GO), including trisaccharide and tetrasaccharide, was performed using a beta-galactosidase in water-hydrophobic solvent mixtures. A maximum GO concentration of 45% (w/w) was attained in a 95% cyclohexane / 5% water mixture from a 55% (w/w) of lactose at 60 degrees C and pH 6.0, while a maximum of 38% GO in aqueous media. GO production decreased with an increase in surfactant concentration. The optimum water content for GO production showed a broad range from 2.5 to 10% (v/v). Solvent properties, such as log P and the dipole moment, had no relation to GO production.