Nucleotide sequencing of the vaccinia virus SalI F DNA fragment identified an open reading frame of 552 amino acids encoding a protein of 63.3 kDa. The deduced amino acid sequence shares 30% identity with S. pombe and S. cerevisiae DNA ligases, with homology strongest near the carboxy terminus and around the lysine residue required for ligase-adenylate formation. Prokaryotic DNA ligases are poorly related to the vaccinia sequence. The initiation codon of the ORF forms part of a late transcriptional initiation sequence TAAATG and is preceded by two overlapping early transcriptional termination signals, TTTTTTTAT. Nonetheless, RNA mapping showed that the ligase gene is transcribed early during infection and the 5' end of the mRNA maps to the TAAATG motif. The possible roles of a DNA ligase in vaccinia virus DNA replication and recombination are discussed.