We found a novel cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. G-825-6. The enzyme was expressed in the culture broth by recombinant Bacillus subtilis KN2 and was purified and characterized. The enzyme named CGTase825-6 showed 95% amino acid sequence identity with a known enzyme beta-/gamma-CGTase from Bacillus firmus/lentus 290-3. However, the product specificity of CGTase825-6 differed from that of beta-/gamma-CGTase. CGTase825-6 produced gamma-cyclodextrin (CD) as the main product, but degradation of gamma-CD was observed with prolonged reaction. The product specificity of the enzyme was positioned between gamma-CGTase produced by Bacillus clarkii 7364 and B. firmus/lentus 290-3 beta-/gamma-CGTase. It showed that the difference of product specificity was dependent on only 28 amino acid residues in 671 residues in CGTase825-6. We compared the amino acid sequence of CGTase825-6 and those of other CGTases and constructed a protein structure model of CGTase825-6. The comparison suggested that the diminished loop (Val138-Asp142) should provide subsite -8 for gamma-CD production and that Asp142 might have an important role in product specificity. CGTase825-6 should be a useful tool to produce gamma-CD and to study the differences of producing mechanisms between gamma-CD and beta-CD.