HEp-2 cells infected with herpes simplex 1 virus contain RNA transcripts capable of forming double-stranded (DS) RNA on annealing. The properties of purified DS RNA were as follows (i) DS RNA is resistant to depolymerization by RNase A or T-1 in 2 times 0.15 M NaCl, plus 0.015 M sodium citrate (SSC) but not 0.1 times SSC or following thermal denaturation. (ii) The Tm of the viral DS RNA was 100 C in 0.1 times SSC. (iii) Undenatured DS RNA does not hybridize with viral DNA: upon denaturation, excess unlabeled RNA drove 50 to 55% of labeled DNA into DNA-RNA hybrid. The kinetics of hybridization indicate that the DS RNA consists of at least two populations of transcripts arising from 29 and 26% of viral DNA and differing 40-fold in molar concentration.