The 4-alpha-glucanotransferase gene (gtpK) of a hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and the nucleotide sequence analysis showed that the gtpK gene is composed of 1,973 bp, which encodes a protein of 653 amino acids with a calculated molecular weight of 76,693 Da. The gtpK was then overexpressed under the control of the lac promoter in Escherichia coli, The enzyme was purified by heat treatment, hydrophobic interaction chromatography, and anion-exchange chromatography. GtpK could react each maltooligosaccharides (from maltose to maltoheptaose) as the effective substrate to form glucose and various maltooligosaccharides containing higher polymeric molecules than the substrate. These results showed that GtpK is a 4-alpha-glucanotransferase which transfers the glucosyl and maltooligosyl units to a new C-4 position in another maltooligosaccharides and glucose. The optimum temperature and pH for the enzyme activity were found to be 100 degrees C and 6-8, respectively, and the enzyme was considerably stable even after heating at 100 degrees C for 30 min. When the deduced amino acid sequence of GtpK was compared with the protein sequences available in the databases, only two significant similar enzymes (alpha-amylases of Pyrococcus furiosus and Dictyoglomus thermophilum exhibited an identity of 68.3% and 36.0%, respectively) were found. Interestingly, only these three homologous enzymes did not preserve the conserved regions of alpha-amylase family, but the other alpha-amylases and 4-alpha-glucanotransferase are classified into the alpha-amylase family since all enzymes have the conserved homologous regions. Therefore, we consider that these enzymes, including GtpK, are classified into a new family of starch-degrading enzymes.