Trehalose biosynthesis genes, treZ, treX and treY, encoding maltooligosyltrehalose trehalohydrolase (TreZ), glycogen debranching enzyme (TreX), and maltooligosyltrehalose synthase (TreY) have been cloned from the thermophilic archaebacterium Sulfolobus acidocaldarius ATCC33909. The amino-acid sequences deduced from treZ, treX and treY are composed of 556, 713 and 720 amino-acid residues, respectively. TreZ and TreY are 33-40% homologous to the corresponding enzymes from Arthrobacter sp. Q36. We have proposed that the biosynthesis of trehalose in Sulfolobus occurs via the actions of the three enzymes encoded by treZXY.