The prokaryotic genomes, for which complete nucleotide sequences are available, always contain at least one RNase H gene, indicating that RNase H is ubiquitous in all prokaryotic cells. Coupled with its unique substrate specificity, the enzyme has been expected to play crucial roles in the biochemical processes associated with DNA replication, gene expression and DNA repair. The physiological role of prokaryotic RNases H, especially of type 1 RNases H, has been extensively studied using Escherichia coli strains that are defective in RNase HI activity or overproduce RNase HI. However, it is not fully understood yet. By contrast, significant progress has been made in this decade in identifying novel RNases H with respect to their biochemical properties and structures, and elucidating catalytic mechanism and substrate recognition mechanism of RNase H. We review the results of these studies.