Earlier reports showed that at least 3 herpes simplex virus 1 proteins were adenylylated and guanylylated in addition to the infected cell proteins (ICPs) 0, 4, 22, and 27. ICP22 and 27 share the amino acid sequence R/PRAP/SR which is also predicted to be in ICPs 4 and 0 and in the products of the HSV-1 genes UL21, UL31, UL47, and UL49. ICPs 0, 4, 22, and 27 are regulatory proteins, UL21 is dispensable for growth in cultured cells, UL31 co-fractionates with the nuclear matrix, and UL47 may interact stoichiometrically with the alpha trans-inducing factor (VP16, the product of the UL48 gene). To avoid bias, the genes encoding the additional nucleotidylylated proteins were mapped, initially by analyses of intertypic recombinant viruses, and subsequently by analyses of the products encoded by the mapped genome domains. We conclude that (i) the products of UL21, UL31, UL47, and UL49 are nucleotidylylated by [alpha-32P]GTP or [alpha-32P]ATP in isolated nuclei, (ii) this modification does not involve the viral protein kinases encoded by the UL13 and US3 gene products and, (iii) UL49 is a virion protein which is labeled in cells with [32P]orthophosphate and also is ADP-ribosylated.