A ribonuclease H which degrades RNA specifically in K N A-DNA hybrids and, moreover, stimulates its homologous DNA-polymerase-primase complex was purified from calf thymus. The enzyme consists of a single polypeptide of molecular mass 78 kDa. It requires divalent cations for activity, and prefers MgZ+ over Mn2+.Ribonuclease H is optimally active at neutral pH and in 75 mM potassium acetate and is strongly sensitive to N-ethylmaleimide.[3H]Poly(rA) . poly(dT), [3H]poly(rC) . poly(dI), and [3H]KNA . MI 3-DNA are degraded to 3 -9-mer oligoribonucleotides with similar kinetics, whereas double-or single-stranded DNA, and double-and single-stranded RNA remain unaffected. The enzyme stimulates in vitro DNA synthesis by the immunoaffinitypurified calf-thymus DNA-polymerase-a -primase complex threefold. When ribonuclease H is present in a threefold molar excess to the polymerase-primase complex, twice as much primer is formed as in the absence of ribonuclease H. Kibonuclease H also stimulates the elongation rate of DNA polymerase L Y by a factor of 2-3. independent of whether primase-primed DNA templates or templates primed with oligonucleotides are used. Our rcsults suggest that this form of ribonuclease H is a likely candidate for a genuine primer-removing enzyme in maininalian cells.DNA replication is a complex process which requires at least 20 -30 different proteins. Central activities for lagging strand synthesis involve a DNA primase for the formation o f short pieces of RNA, typically 5 -10 nucleotides long. which serve as primers for the replicative DNA polymerase (for a review see 11, 21). In eukaryotic organisnis RNA primer formation and DNA synthesis are performed by a multisubunit complex, named DNA polymerase-or -primase complex (for a review see [3]). The removal of RNA primers is a prerequisite for the subsequent sealing of Okazaki fragments by DNA ligation. Most likely, a ribonuclease specific for the RNA strand of a DNA-RNA hybrid, ribonuclease H (RNase H; EC 3.1.4.34), is responsible for primer removal. Such an enzyme was first detected by Skin and Hausen in calf thymus 141. Since then, the puritication and characterization of several different forms of RNase H from many organisms has been reported (for a review see [S]). However, until now it was not clear which of the known forms of RNase H were involved in the process of DNA replication.Our laboratory is concerned with the identification and isolation of proteins involved in DNA replication in calf thymus cells in order to reconstitute a replication complex in vitro. These studies began with the isolation of the DNA polymerase cc [6, 71 and were continued by the isolation and Corresponderrce to F. Grosse, Abteilung Chemie.