The enzyme RNase H (EC 3.1.26.4) is a ribonuclease that cleaves the 3'-O-P-bond of RNA in a DNA/RNA duplex to produce 3'-hydroxyl and 5'-phosphate terminated products. RNase H is an non-specific endonuclease and catalyzes the cleavage of RNA via an hydrolytic mechanism, aided by a enzyme-bound divalent metal ion. In contrast to other ribonucleases, such as RNase A or RNase T1 , RNase H leaves a 3'-phosphorylated product.

Members of the RNase H family can be found in nearly all organisms, from archaea and prokaryota to eukaryota. In eukaryotic DNA replication, RNAse H is responsible for cutting out the RNA primer, allowing completion of the newly synthesized DNA.

Retroviral RNase H, a part of the viral reverse transcriptase enzyme, is an important pharmaceutical target, as it is absolutely necessary for the proliferation of retroviruses, such as HIV. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS. As of 2004, there are no RNase H inhibitors in clinical trials, though some approaches employing DNA aptamers are in the preclinical stage.

In a molecular biology laboratory, as RNase H specifically degrades the RNA in RNA:DNA hybrids and will not degrade DNA or unhybridized RNA, it is commonly used to destroy the RNA template after first-strand complementary DNA (cDNA) synthesis by reverse transcription, as well as procedures such as nuclease protection assays . RNase H can also be used to degrade specific RNA strands when the cDNA oligo is hybridized, such as the removal of the poly(A) tail from mRNA hybridized to oligo(dT). To terminate the reaction, a chelator, such as EDTA, is often added to sequester the required metal ions in the reaction mixture.