Recently we have shown that the major isoform of RNase H in human cells, RNase H1, is able to cleave DNA substrates containing a single RNA-DNA base pair, an activity which appears to be involved in an excision repair system for the removal of ribose residues misincorporated into DNA. In the present work we have further characterized the substrate specificity of the enzyme. DNA substrates containing all four ribonucleotides are cleaved by the enzyme. A RNA-DNA base pair is not required for substrate recognition. RNA residues present within a mismatch or in a RNA-RNA base pair are also cleaved. The principal structural feature for recognition by the enzyme may simply be the presence of the 2'-OH group of the ribose residue adjacent to the cleavage site.