Ribonuclease T₁ (EC 3.1.27.3, guanyloribonuclease, Aspergillus oryzae ribonuclease, RNase N1, RNase N2, ribonuclease N3, ribonuclease U1, ribonuclease F1, ribonuclease Ch, ribonuclease PP1, ribonuclease SA, RNase F1, ribonuclease C2, binase, RNase Sa, guanyl-specific RNase, RNase G, RNase T₁, ribonuclease guaninenucleotido-2'-transferase (cyclizing), ribonuclease N3, ribonuclease N1) is a fungal endonuclease that cleaves single-stranded RNA after guanine residues, i.e., on their 3' end; the most commonly studied form of this enzyme is the version found in the mold Aspergillus oryzae. Owing to its specificity for guanine, RNase T₁ is often used to digest denatured RNA prior to sequencing. Similar to other ribonucleases such as barnase and RNase A, ribonuclease T₁ has been popular for folding studies.

Structurally, ribonuclease T₁ is a small α+β protein (104 amino acids) with a four-stranded, antiparallel beta sheet covering a long alpha helix (almost five turns). RNase T₁ has two disulfide bonds, Cys2-Cys10 and Cys6-Cys103, of which the latter contributes more to its folding stability; complete reduction of both disulfides usually unfolds the protein, although its folding can be rescued with high salt concentrations.

RNase T₁ also has four prolines, two of which (Pro39 and Pro55) have cis isomers of their X-Pro peptide bonds. Nonnative isomers of these prolines can retard conformational folding dramatically, folding on a characteristic time scale of 7,000 seconds (almost two hours) at 10 °C and pH 5.