Nucleotide sequencing of a 1,300-base-pair vaccinia virus DNA segment previously shown to contain a thymidine kinase (TK) gene revealed an uninterrupted reading frame of 177 codons capable of producing a polypeptide with a molecular weight of 20,102. Mapping of the TK mRNA by primer extension indicated a unique 5' end that precedes the initiation codon by only six nucleotides. Multiple 3' ends within a 10-nucleotide region, about 30 nucleotides beyond the termination codon, were located by nuclease digestion of DNA-RNA hybrids, and the length of the TK transcript, exclusive of polyadenylate, was estimated to be approximately 570 nucleotides. The region preceding the TK mRNA start site is extremely A + T rich and has sequence homologies with three other early genes. Genetic information is so compressed in this region of the DNA that the putative transcriptional regulatory sequence of the TK gene overlaps the coding sequence of a late gene. Only nine nucleotides separate the termination codon of the late gene from the initiation codon of the TK gene. Downstream, 66 nucleotides separate the TK termination codon from the apparent initiation codon of another early gene. The nature of three independent TK- mutants was revealed by nucleotide sequencing. Each has a nucleotide reiteration leading to a +1 frameshift and a nonsense codon downstream. The location of one frameshift mutation provided evidence that the first ATG is used for initiation of protein synthesis.