A hyperthermophilic archaeon, Pyrococcus sp. KOD1, produces extracellular thermostable alpha-amylase (ApkA). The apkA gene was cloned and expressed in Escherichia coli. Nucleotide sequence analysis showed that the apkA gene is composed of 1,383 bp (461 amino acid residues) and contains a signal sequence (26 N-terminal amino acid residues). The molecular weight of the mature enzyme was estimated to be 49,456 Da with 435 amino acid residues. The deduced amino acid sequence of the mature enzyme was compared with those of alpha-amylase and other starch-degrading enzymes from a variety of organisms. The overall homology to other amylases was not high (below 40%); however, high homology was found in the four conserved regions of the alpha-amylase family. The cloned apkA was overexpressed using the T7 RNA polymerase expression system. The enzyme was purified by ammonium sulfate fractionation, heat treatment, anion-exchange chromatography, and hydrophobic interaction chromatography. The optimum temperature and pH for the enzyme activity were found to be 90 degrees C and pH 6.5, and the enzyme was considerably stable even after heating at 90 degrees C for 60 min. The thermostability of the enzyme was enhanced in the presence of 2 mM Ca2+. ApkA hydrolyzed soluble starch and glycogen to form maltose and maltotriose as the major products, and very weakly hydrolyzed pullulan to form panose or isopanose, suggesting cleavage at the alpha-1, 4-linkage of pullulan. The steady-state kinetic constants were determined for various substrates, and ApkA was found to show high affinity to highly branched polysaccharides such as glycogen.