1. A ribonuclease (H.S. RNase) was extracted from horse submaxillary gland with isotonic KC1 and purified about 15, 000-fold with an over-all yield of about 14%. 2. The enzyme was free of DNase [EC 3.1.4.5], nonspecific phosphodiesterase [EC 3.1.4.1] and phosphomonoesterase [EC 3.1.3.1] activities, and appeared homogeneous on ultracentrifugation, paper electrophoresis, CM-cellulose column chromatography and gel filtration. 3. The mode of action of H.S. RNase on RNA was found to be similar to that of RNase A* [ribonucleate pyrimidinenucleotido-2′-transferase (cyclizing), EC 2.7.7.16]. However, the base specificities of H.S. RNase and RNase A differed the former exhibiting little if any preference for cytosine over uracil in the degradation of 2′, 3′-cyclic nucleotides and RNA at pH 8.3. 4. H.S. RNase was found to differ from RNase A in molecular weight estimated by a Sephadex gel filtration method (H.S. RNase: ca. 24, 000), specific activity (H.S. RNase: RNase A=1: 2), elution position from a CM-cellulose column, and resistance to inhibition by heparin. Small but significant differences were also observed in the isoelectric points and pH optima of these enzymes. 5. H.S. RNase is very similar to RNase A in its heat stability and inhibition by Zn2+, Cd2+, Cu2+ and Hg2+.