Thermostable RNase H is an endoribonuclease that is functional at high temperatures and selectively hydrolyzes the phosphodiester bonds of an RNA strand in an RNA:DNA hybrid molecule, while leaving the DNA strand intact. In addition, Thermostable RNase H does not degrade single- or double-stranded RNA or DNA. While the Thermostable and E. coli RNase H homologs both exhibit these same endoribonucleolytic properties, the thermostable enzyme displays activity at higher temperatures with optimal activity above 65°C. These properties allow use in applications where higher assay temperatures are desired.