The deoxyuridine triphosphate nucleotidohydrolase (dUTPase) activity which is induced in KB cells infected with herpes simplex virus type 2 (HSV-2) and expressed in biochemically transformed HeLa cells was separated from the host dUTPase activity using either gel filtration or chromatofocusing chromatography. The HSV-2-induced dUTPase differed from the host dUTPase activity in its molecular weight (53000 versus 46000) and isoelectric point (pI 5 X 9 versus 6 X 4). However, it was similar to the host enzyme in that only dUTP served as a substrate for the enzyme.