Two forms of ribonuclease H (RNase H) have been identified both in uninfected and Herpes Simplex virus (HSV-)infected BHK cells. Identical RNase H species were detected in control- as well as in infected cells. RNase H I and II have not been found to be associated both with host cell DNA polymerase alpha and beta and HSV-induced DNA polymerase. Infection of BHK cells with HSV type 1 does not lead to a pronounced alteration of RNase H II activity but to an increase (3-fold) of the extractable RNase H I activity. RNase H I activity increases to a maximum between 8-10 hours p.i.; the bulk of HSV-DNA synthesis occurs between 6-8 hours p.i. From these experiments we draw the preliminary conclusion that RNase H I is involved in the degradation of the RNA primer which is covalently linked to newly synthesized HSV-DNA strands.