We have investigated the organization of transcriptional units within a 9000 bp segment of the terminally repeated region of the DNA genome of vaccinia virus, which uses its own enzyme system to synthesize mRNA within the cytoplasm of infected cells. RNA splicing, which has been demonstrated for DNA viruses that replicate within the nucleus of infected cells, does not appear to be involved in the formation of these first vaccinia virus mRNAs to be examined. Three immediate early mRNAs, approximately 1050, 600 and 1100 nucleotides long, were mapped between 3.21 and 4.24, 6.54 and 7.16, and 7.20 and 8.23 kb from the end of the genome, respectively. The direction of transcription was toward the end of the genome for the two larger mRNAs and in the opposite direction for the smallest one. Additional minor RNAs, which were larger in size, were mapped between and to the same DNA strand as the mRNAs of 1050 and 1100 nucleotides. No evidence for interrupted genes was obtained by nuclease S1 analysis after hybridization of RNA to labeled DNA. In addition, the 5' ends of the mRNAs, which were specifically labeled by in vitro capping, hybridized to DNA adjacent to the body of the message.