A site-specific endonuclease has been isolated from Thermus thermophilus HB8 and named TthHB8I. It recognizes the same sequences as TaqI from Thermus aquaticus YT-1 does. The amount of TthHB8I in the cells was comparable to that of TaqI. T. thermophilus HB8 has an advantage over T. aquaticus YT-1 for preparation of a TaqI-like enzyme since it is easier to obtain T. thermophilus HB8 cells in quantity.