Analysis of nuclei isolated from herpes simplex virus (HSV)-infected cells by electrophoresis in polyacrylamide gels showed that only virion-associated DNA molecules migrated into the gels. The viral DNA molecules, which do not migrate in the gels, are the precursors for the mature viral DNA. Centrifugation of deoxycholate-treated infected nuclei in sucrose gradients revealed that most of the viral DNA co-sedimented with the cellular DNA. The DNA-dependent RNA polymerase activity also co-sedimented with the viral and cellular DNA. Incubation of nuclei isolated from HSV-infected cells, under in vitro conditions that support DNA synthesis, resulted in the synthesis of viral molecules of low molecular weight. Under the same conditions, nuclei from Burkitt lymphoblasts did not synthesize DNA. The nature of the association between cellular DNA and EBV DNA was studied by hybridization with EBV cRNA. EBV-specific sequences were found in the lymphoblast DNA; they banded at a density of 1.707 g/cm3. Some of the viral mRNA transcribed in HSV-infected nuclei is symmetrically transcribed from HSV DNA. The symmetrical portions of the RNA are removed, since poly (A)-containing mRNA molecules lack homologous sequences. Most of the RNA synthesized in HSV-infected nuclei can be released after incubation of the nuclei in vitro in the presence of ATP. The released RNA consists of poly(A)plus and poly (A)-minus molecules. The mechanism of RNA transport from the nuclei still remains to be studied.