beta-Galactosidase catalyzed beta-galactosylation not only of a hydroxyl group but also of a thiol group in the condensation reaction of D-galactose and 2-mercaptoethanol. The thio-galactosylation product was confirmed as 2-hydroxyethyl S-beta-D-galactoside on the bases of fast atom bombardment mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectorometry. Aspergillus oryzae beta-galactosidase hydrolyzed p-nitrophenyl S-beta-D-galactoside most rapidly among several beta-galactosidases and produced the thio-galactosylation product most efficiently. The Penicillim multicolor enzyme was as effective as the A. oryzae enzyme. However the enzymes from Escherichia coli, Saccharomyces fragilis, Kluyveromyces lactis, and Bacillus circulans galactosylated hydroxyl groups predominantly to produce O-galactoside. The thio-galactoside was synthesized most effectively at a 2-mercaptoethanol concentration of about 1.25 M. Galactose concentration at 0.8-2.8 M did not affect the synthetic yield of the thiogalactoside so greatly.