Less purified fractions of ribonuclease H IIa activity of calf thymus display divalent cation-dependent ribonuclease H activity and divalent cation-independent ribonuclease activity. Because the ratio of the two enzyme activities does not change during successive chromatographic procedures, we suggest that ribonuclease H IIa activity is indeed able to degrade both ssRNA and the RNA moiety of RNA.DNA-hybrids. Ribonuclease H IIa activity can therefore be differentiated from calf thymus ribonuclease H I and H IIb by its lack of ribonuclease H specificity. The native molecular mass of ribonuclease H IIa activity is between 23 and 28 kDa. Under denaturing conditions a 23 kDa-protein band copurifies with the enzyme activity suggesting that this enzyme is monomeric.