A cDNA clone encoding ubiquitin-activating enzyme E1 has been isolated from a rabbit heart cDNA library and sequenced. The 3.485 kb cDNA contains an open reading frame of 1058 amino acid residues which predicts a protein of approx. 118 kDa. The deduced protein sequence exhibits a very high homology to other ubiquitin-activating enzymes identified in a variety of organisms. Northern blot analysis reveals a single transcript of approx. 3.5 kb in all the rabbit tissues examined. The entire coding region of the rabbit E1 cDNA has been expressed as a his-tagged protein. The recombinant protein has been verified by its ability to cross-react with anti-human E1 antibodies. Ubiquitin thiolester assay shows that the recombinant rabbit E1 protein is functional.