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Summary The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells., Graphical Abstract Highlights ► Ribonucleotides are the most common nucleotide base lesion in the mouse genome ► RNase H2 is a key genome surveillance enzyme required for removal of nucleotides ► RNase H2 is essential for mammalian development ► Without RNase H2, cells exhibit genome instability and p53 pathway activation, DNA polymerases can incorporate more than a million ribonucleotides into replicating mouse DNA in each cell, making ribonucleotides the most abundant kind of DNA lesion. RNase H2, which removes this “damage,” has an essential role in genome surveillance and is required for embryonic development.