In an in vitro system containing enzymes extracted from vaccinia virions, transcription of the vaccinia growth factor gene terminated approximately 50 base pairs downstream of a thymidine-rich sequence. Deletion mutagenesis suggested the presence of two tandem termination signals. The signal was identified by replacing the 3' end of the gene with the oligonucleotide AATTTTTAT that induced downstream termination. Further analysis of the transcripts formed with a series of templates containing 16 related synthetic oligonucleotides established the minimum functional termination signal as TTTTTNT, in which N represents any nucleotide. Termination efficiency may be increased, however, by the presence of an adenosine preceding the thymidine cluster. The general use of this signal at early times in infection but not at late times is supported by a survey of vaccinia virus gene sequences.