Abstract The 140-kDa RR1 protein from both herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) functions as the large subunit of viral ribonucleotide reductase (RR) and contains an intrinsic serine/threonine protein kinase (PK) activity at the unique NH 2-terminal domain. This PK activity is capable of autophosphorylation and reported to transphosphorylate histone and calmodulin by some groups but not by others. It has been suggested that the lack of consensus in the finding of transphosphorylation activity with RR1 protein may be due to HSV strain variations used in different laboratories. In the present study, we have attempted to resolve this issue by immunospecifically isolating the 140-kDa RR1 protein from four different strains of HSV-1 including F, KOS, HF, and MP and three different strains of HSV-2 including G, 333, and MS and subjecting them to immunocomplex kinase assays. In PK assays autophosphorylation of 140-kDa RR1 was readily observed in protein immunopurified from all HSV-1 and HSV-2 strains used in this study. However, using the same assay no transphosphorylation activity was observed with either the 38-kDa RR2 protein present in immunocomplex along with the RR1, or with histone, when added as an exogenous PK substrate. This conclusion is further supported by the evidence that a commercial preparation of PK (protein kinase catalytic subunit from bovine heart) readily phosphorylated histone under the conditions used for RR1 immunocomplex kinase assay. These results show that the 140-kDa RR1 protein contains an autokinase activity but it is incapable of transphosphorylating heterologous substrates such as histone. In addition, we show that the RR enzyme complex (140 and 38-kDa proteins) is associated with purified HSV-2 virions.