The enzyme with beta-galactosidase activity from Sulfolobus solfataricus strain MT-4, like other enzymes of this type isolated from thermophilic sources, has broad specificity for beta-D-gluco-, fuco- and galacto-sides. The beta-galactosidase activity was purified by a new procedure that improved yields (44%) and final specific activity (182 units mg-1 at 75 degrees C using chromogenic beta-D-galactoside as substrate). The enzyme hydrolysed a large number of beta-linked glycoside dimers and oligomers; chromogenic beta-glucosides and beta-fucosides are the preferred substrates, and kinetic analysis indicated that they bind to a common catalytic site. The order of catalytic efficiency was beta 1-3 > beta 1-4 > beta 1-6 and cellotetraose > cellotriose > cellobiose for glucose dimers and oliogomers respectively. The cleavage occurred at the non-reducing end of the oligosaccharide, and the enzyme showed noticeable specificity also for the aglycone part of substrates. From these results the enzyme from S. solfataricus strain MT-4 is defined as a true glycosyl hydrolase with remarkable exo-glucosidase activity and it is designated S beta-gly.