Abstract: A substantially pure preparation of thermostable RNase H is disclosed. In one preferable form of the invention, the RNase H is capable of biological activity after incubation at temperatures equal to or greater than 70.degree. C. for at least ten minutes. In another preferable form of the invention, the purification is from Thermus thermophilus or a closely related organism. The present invention is also a method of digesting RNA polymers that are in duplex form with a DNA molecule. The method comprises exposing the duplex to the isolated thermostable RNase H.