RNase H is an enzyme that specifically cleaves RNA hybridized to DNA. The enzyme is ubiquitously present in various organisms. Single bacterial and eucaryotic tells often contain two RNases H, whereas single archaeal cells contain only one. To determine whether there is a physiologicaI significance in the ubiquity and multiplicity of the enzyme, and whether all enzymes are evolutionarily diverged from a common ancestor, we carried out phylogenetic analyses of the RNase H sequences. In this report, we demonstrated that RNases H are classified into two major families, Type 1 and Type 2 RNases H, of which only the Type 2 enzymes are present in all Living organisms, including bacteria, archaea, and eucaryotes, suggesting that they represent an ancestral form of RNases H. Based on this information, we discuss the evolutionary relationships and possible cellular functions of RNases H.