Abstract Treatment of calf serum at 60°C and pH 3.5 followed by chromatography on carboxymethyl (CM) cellulose resulted in the separation of two major peaks of alkaline RNAase activity. One was eluted from CM-cellulose at 0.075 M KCl with an overall purification of 5400-fold and the other was eluted at 0.25 M KCl with a 6700-fold purification. The RNAase eluted from CM-cellulose at 0.075 M KCl was almost completely inhibited by anti-RNAase A serum and by the endogenous RNAase inhibitor and a 33% inhibition was observed in the presence of 5 mM MgCl 2. This enzyme seems to be similar or identical to RNAase A. The other RNAase, eluted from CM-cellulose at 0.25 M KCl was not inhibited by anti-RNAase A or 5 mM MgCl 2 and was much less sensitive to the endogenous inhibitor. Both enzymes degraded RNA endonucleolytically and the nucleoside monophosphates obtained after partial hydrolysis of RNA by the two serum RNAases were primarily 2′- or 3′-CMP and 2′- or 3′-UMP. Poly(A), native DNA and denatured DNA were degraded slowly or not at all. The RNAase A-like enzyme degraded poly(C) at a significantly faster rate, and poly(U) at a slower rate, than RNA. However, the other serum RNAase was more active with poly(U) than with RNA and almost inactive with poly(C) as the substrate.