Fractionation of polyadenylated RNA from cells infected with herpes simplex virus by affinity chromatography on columns of poly (U) immobilized on glass-fiber filters yielded three major classes of RNA-containing poly(A) chains with average lengths of 30, 50, and 155 adenylate residues [poly(A)30, poly(A)50, poly(A)155]. In contrast, nitrocellulose membranes bound predominantly a fraction of RNA containing poly(A)155. The distribution of cytoplasmic RNA in the three classes was found to be independent of the labeling interval, ranging from 10 min to 6 h. Cytoplasmic poly(A) RNA consisted mainly (57 to 68%) of the poly(A)155 class; this was also the major class (68%) of polyadenylated RNA found in polyribosomes. Nuclear poly(A) RNA consisted largely (42 to 50%) of poly(A)30 class, whereas high-molecular-weight nuclear RNA sedimenting at greater than 45S contained almost exclusively the poly(A)30 tracts. Hybridization experiments involving unlabeled RNA and labeled viral DNA demonstrated the presence of viral RNA sequences complementary to approximately 40% of viral DNA in all polyadenylated RNA classes. Inasmuch as unfractionated cytoplasmic RNA arises from approximately 40% of the viral DNA, we conclude that most, if not all, viral RNA species present in the cytoplasm are adenylated. In contrast to these results, only a fraction of poly(A)155 RNA, complementary to 21% of viral DNA, bound to nitrocellulose filters. The selective binding of poly(A)155 sequences to nitrocellulose filters might be related to its secondary structure, since transcripts homologous to 40% of viral DNA bind to nitrocellulose membranes, provided the RNA is denatured prior to filtration. The data suggest that poly(A) tracts arise by at least two separate steps. The first involves the appearance of poly(A)30 tracts in the high-molecular-weight nuclear transcripts. The second involves polyadenylation to ply(A)50 and poly(A)155 RNA classes concomitant with processing and transport to the cytoplasm.