The sequence complexity and relative abundance of vaccinia virus mRNA's, synthesized in vivo and in vitro, have been measured by DNA-RNA hybridization. Up to 42% of [3H]thymidine-labeled virus DNA can be protected from digestion with nuclease S1, a single-strand specific nuclease, after annealing to excess polyadenylylated mRNA obtained at 7 h after infection. In contrast, only 26% of vaccinia virus DNA is protected when hybridized to polyadenylylated RNA obtained at 2 h after infection in the presence of an inhibitor of DNA synthesis. That the 94 kilobases transcribed early are a subset of the 152 kilobases present late was suggested by hybridization of DNA with a mixture of early and late RNAs. Some control of transcription is lost when virus purified by procedures that include sonic treatment is used for infection since under these conditions similar proportions of DNA are protected by either excess early or late RNA. Excess RNA, synthesized in vitro by enzymes within purified vaccinia virus particles, hybridized to approximately the same fraction of the DNA as did RNA present at late times in vivo. A second type of transcriptional control was demonstrated by kinetic analysis of the hybridization of polyadenylylated RNA to labeled DNA. With virion DNA used as the probe, a single abundance class for early RNA, two classes differing 11-fold in abundance for late RNA, and two classes differing 43-fold in abundance for in vitro RNA were found. To be able to detect high-abundance RNAs of very low sequence complexity, labeled complementary DNA probes to early, late, and in vitro polyadenylylated RNA were used. Evidence that, at late times, RNAs totaling 9 kilobases of sequence complexity are present 40 to 500 times more frequently than the bulk of the virus-specific RNA was obtained. In contrast, the highest abundance class of RNA present at 2 h after infection corresponded to 7 kilobases present in only a 13-fold molar excess over the majority of virus-specific sequences. RNA synthesized in vitro was found to contain a small amount of sequence information, approximately 2 kilobases, which occurred 150 times more frequently than the majority of viral sequences. Studies using hybridization of viral DNA to labeled complementary DNA probes also suggested that 52 to 59% of the polyadenylylated RNA present at 2 h after infection and 82 to 92% of that at 7 h are virus specific.