A 3382 bp fragment containing a gene for a DNA ligase from the extremely thermophilic, acidophilic, and facultatively anaerobic archaeon (archaebacterium) Desulfurolobus ambivalens was cloned and sequenced. The deduced amino acid sequence (600 amino acids, 67619 molecular weight) showed 30-34% sequence identity with the ATP-dependent eucaryal (eukaryotic) DNA ligases of Schizosaccharomyces pombe, Saccharomyces cerevisiae, the human DNA ligase I, and with the Vaccinia DNA ligase. Distant similarity to the DNA ligases from the bacteriophages T3, T4, T6, T7 and the African swine fever virus was found, whereas no similarities were detectable to the NAD-dependent DNA ligases from the bacteria (eubacteria) Escherichia coli and Thermus thermophilus, to the ATP-dependent RNA-ligase of bacteriophage T4, and to the tRNA-Ligase from S.cerevisiae. A detailed comparison of the phylogenetic relationship of the amino acid sequences of all known DNA and RNA ligases is presented including a complete alignment of the ATP-dependent DNA ligases. The in vivo-transcription initiation and termination sites of the D.ambivalens gene were mapped. The calculated transcript length was 1904-1911 nt.