The Bacillus sp. SAM1606 alpha-glucosidase with a broad substrate specificity is the only known alpha-glucosidase that can hydrolyze alpha,alpha'-trehalose efficiently. The enzyme exhibits a very high sequence similarity to the oligo-1,6-glucosidases (O16G) of Bacillus thermoglucosidasius and Bacillus cereus which cannot act on trehalose. These three enzymes share 80% identical residues within the conserved regions (CR), which have been suggested to be located near or at the active site of the alpha-amylase family enzymes. To identify by site-specific mutagenesis the critical residues that determine the broad substrate specificity of the SAM1606 enzyme we compared the CR sequences of these three glucosidases and selected five targets to be mutagenized in SAM1606 alpha-glucosidase, Met76, Arg81, Ala116, Gly273, and Thr342. These residues have been specifically replaced by in vitro mutagenesis with Asn, Ser, Val, Pro, and Asn, respectively, as in the Bacillus O16G. The 12 mutant enzymes with single and multiple substitutions were expressed and characterized kinetically. The results showed that the 5-fold mutation virtually abolished the affinity of the enzyme for alpha, alpha'-trehalose, whereas the specificity constant for the hydrolysis of isomaltose, a good substrate for both the SAM1606 enzyme and O16G, remained essentially unchanged upon the mutation. This loss in affinity for trehalose was critically governed by a Gly273 --> Pro substitution, whose effect was specifically enhanced by the Thr342 --> Asn substitution in the 5-fold and quadruple mutants. These results provide evidence for the differential roles of the amino acid residues in the CR in determining the substrate specificity of the alpha-glucosidase.