Bacillus flavothermus alpha-amylase was purified to homogeneity using a combination of ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The enzyme displayed maximal activity on starch at pH 5.5-6.0 and 60 degrees C and had an isoelectric point of 8.4 and a K-m of 2.2 mg ml(-1). Diethyl pyrocarbonate inactivated the amylase at pH 5.5 and 20 degrees C in a monomolecular reaction with a second-order rate constant of 250 M(-1) min(-1). The influence of pH on the rate of inactivation suggested the participation of a residue with a pK(a) of 6.7. Spectrophotometric studies and reactivation in the presence of hydroxylamine suggested the modification of histidine(s). A single histidine residue appeared to be essential and the substrate afforded complete protection indicating its location at the active site of the enzyme.