We have demonstrated by primer elongation and cap analysis that mature vaccinia virus late transcripts are discontinuously synthesized. We have shown that RNA transcripts from a translocated 11K and from the authentic 11K and 4b late promoters are extended by approximately 35 nucleotides beyond the "start site" determined by S1 mapping using vaccinia genomic DNA as a probe. Sequencing of the RNA and of the first strand cDNA reveal that a homopolymeric poly(A) sequence is linked to the 5' terminus of the RNA transcripts. S1 mapping of RNA transcripts with a DNA probe containing an A-stretch, replacing promoter sequences upstream of position -1, confirms the existence of a poly(A) leader of approximately 35 A-residues.