Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable ␣-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for ␣-linked galactosides, which are optimally hydrolyzed at pH 5 and 90°C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable ␣-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.␣-Galactosidases (␣-Gals) (EC 3.2.1.22) are a widespread class of enzymes that liberate galactose from the nonreducing end of sugars. In bacteria, yeasts, and fungi, these enzymes are usually involved in the degradation of various plant saccharides, which can then serve as a carbon and energy source for growth. Plants synthesize ␣-galactosides such as raffinose and stachyose as the major energy storage molecules in leaves, roots, and tubers. Moreover, these oligosaccharides have been associated with cold and desiccation tolerance of seeds (28). Both raffinose and stachyose are produced by two specialized synthases (raffinose synthase [EC 2.4.1.82] and stachyose synthase [EC 2.4.1.67]) that use galactinol as a galactosyl donor (39). The oligosaccharides are degraded during seed germination by the action of two distinct types of ␣-Gals that differ in their optimal pH of catalysis. While the acid ␣-Gal type is most likely active in the acidic environment of the vacuole and the apoplasm, the alkaline ␣-Gal type probably catalyzes galactose release in the more neutral or alkaline cytoplasm (4,17,31). Mammals express an ␣-Gal and an ␣-N-acetylgalactosaminidase (␣-NAGal) in lysosomal bodies to degrade glycolipids, glycoproteins, and oligosaccharides. In humans, mutations in