Nuclei isolated from herpes simplex virus (HSV)-infected KB cells actively synthesize HSV RNA in vitro; the RNA can be hybridized with HSV DNA or nuclear RNA from HSV-infected cells. Nascent RNA molecules labeled in vivo with 32PO4 were elongated, utilizing the nuclear system to incorporate Hg-CTP at their 3' ends, and then isolated on an affinity column. Hybridization of isolated nascent RNA molecules showed that greater than 50% of them were HSV specific and that more than 25% were self-complementary.