The gene encoding for an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) was sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (1686 base pairs) corresponded to a protein of 562 amino acid residues with a Mr of 66,502. Its predicted amino acid composition, Mr, and N-terminal sequence of 12 residues were consistent with those determined for B. thermoglucosidasius oligo-1,6-glucosidase. The deduced sequence of the enzyme was 72% homologous to that of a thermolabile oligo-1,6-glucosidase (558 residues) from Bacillus cereus ATCC7064 (mesophile). B. cereus oligo-1,6-glucosidase contained 19 prolines. Eighteen of these were conserved at the equivalent positions of B. thermoglucosidasius oligo-1,6-glucosidase. This enzyme contained 14 extra prolines besides the conservative prolines. The majority of extra prolines was replaced by polar or charged residues (Glu, Thr, or Lys) in B. cereus oligo-1,6-glucosidase. The extra prolines were responsible for the difference in thermostability between these two enzymes. We suggested that 11 of the extra prolines in B. thermoglucosidasius oligo-1,6-glucosidase occur in beta-turns or in coils within the loops binding adjacent secondary structures.