Themoactinomyces vulgaris R-47 produces two a-amylases, TVA I and TVA II, both of which hydrolyze pullulan and cyclodextrins. Site-directed mutagenic and X-ray crystallographic studies ofboth enzymes were carried out. The putative catalytic residues, Asp325, G1u354, and Asp421 of TVA II were modified. The mutated enzymes retained a trace of activity, less than 0.01% of the wild-type enzyme, and the action pattern of D421N enzyme was different from wild-type and othermutated TVA II. The crystal structures of the mutated TVA II complexed with β-cyclodextrin were determined. TVA II has domains A, B and C, like other a-amylases, and also has domain N, which is not commonly found in α-amylase family enzymes. A β-cyclodextrin was bound to domain A, and was also located close to domain B. Crystals of TVA I were obtained by the vapor diffusion method in the presence of polyethylene glycol. Like TVA II, TVA Iwas composed of domain N, A, B, and C. Comparing the domains of TVA I and TVA II, the structure and position of domain N were the most different. To investigate the roles of domain N further, domains N of TVA I and TVA II were truncated, and the activities of these enzymes were drastically decreased.