Denaturated 3H-thymidine-labeled vaccinia virus DNA was hybridized with an excess of "late" virus-specific RNA isolated from virus-infected chick embryo cell cultures 8 hours postinfection. The percentage of DNA converted into DNA-RNA hybrid under these conditions never exceeded 50%. If the RNA preparation had been self-annealed prior to hybridization, the percentage was decreased slightly. On the other hand, if the self-annealed RNA had been treated with RN-ase and the double-stranded DNA-RNA hybrids had been denaturated, they became capable of converting into hybrids at least 68% of the labeled DNA. These data indicate that transcription of both DNA strands occurs in a large portion (over 68%) of vaccinia virus genome.