A thermostable alpha-amylase gene of a novel hyperthermophilic archaeon, Thermococcus profundus, was cloned and expressed in Escherichia coli. E. coli cells transformed with the gene overproduced the amylase to a level as much as 155.5-fold higher than that produced by T. profundus. Up to 45% of the total activity was secreted into periplasmic fraction and culture filtrate. The nucleotide sequence of the gene displayed an open reading frame of 1,203 bp encoding a 401-amino acid protein, which was composed of a 26-amino acid signal peptide and a 375-amino acid mature enzyme. Analysis of the deduced amino acid sequence revealed that four homologous regions common to amylases were conserved in the alpha-amylase. The molecular mass of the mature enzyme was calculated to be 43,281 Da, which is consistent with the value obtained from results of SDS-polyacrylamide gel electrophoresis of the purified enzyme, The amylase purified to homogeneity from the E. coli transformant exhibited properties identical to those of native amylase from T. profundus in terms of thermoactivity, pH profile, and the mode of action on various substrates. The amylase did not hydrolyze alpha-1,6-glucosidic linkages.