X-ray diffraction analysis (at 2.1-Å resolution) of an acid α-amylase from Aspergillus niger allowed a detailed description of the stereochemistry of the calcium-binding sites. The primary site (which is essential in maintaining proper folding around the active site) contains a tightly bound Ca2+ with an unusually high number of eight ligands (051 and 052 of Aspl75, 05 of Asnl21, main-chain carbonyl oxygens of Glu 162 and Glu210, and three water molecules). A secondary binding site was identified at the bottom of the substrate binding cleft; it involves the residues presumed to play a catalytic role (Asp206 and Glu230). This explains the inhibitory effect of calcium observed at higher concentrations. Neutral Aspergillus oryzae (TAKA) α-amylase was also refined in a new crystal at 2.1-Å resolution. The structure of this homologous (over 80%) enzyme and additional kinetic studies support all the structural conclusions regarding both calcium-binding sites.