The gene (inuA) encoding exo-inulinase (EC 3.2.1.80) was cloned from the thermophilic Geobacillus stearothermophilus (Bacillus stearothermophilus) KP 1289 growing at between 41degreesC and 69degreesC. The inuA gene consisted of 1,482 bp encoding a protein of 493 amino acids. The deduced polypeptide of molecular mass (M) 56,744 Da showed strong sequence similarity to Pseudomonas mucidolens exo-inulinase, Bacillus subtilis levanase, Paenibacillus polymyxa (Bacillus polymyxa) fructosyltransferase, and so on, indicating that the enzyme belonged to glycosyl hydrolase family 32. The M of the purified exo-inulinase, expressed in Escherichia coli HB101, was estimated as approximately 54,000 Da by both SDS-PAGE and gel filtration. These results suggested that the active form of the enzyme is a monomer. The enzyme was active between 30 and 75degreesC with an optimum at 60degreesC. The properties were identical to those of the native enzyme. Additionally, for the first time for a prokaryotic GH32 protein, crystals of the recombinant enzyme were obtained.