The gene coding for the guanylyltransferase of African swine fever virus has been identified and sequenced. The gene, designated NP868R, is located within fragments EcoRI N' and D of the virus genome (BA71V strain) and encodes a protein with a predicted molecular mass of 99.9 kDa that shares significant similarity with the large subunit of both vaccinia and Shope fibroma virus capping enzymes, with percentages of identity of 20.6 and 21.8%, respectively. A protein of 95 kDa was induced in Escherichia coli cells transformed with a recombinant plasmid carrying the NP868R gene. The E. coli expressed protein, as well as a protein of the same molecular weight present in African swine fever virus particles, form a covalent complex with GTP that can be reversed by pyrophosphate, two characteristic reactions of guanylyltransferases. An examination of the amino acid sequences of the African swine fever virus, poxvirus, and yeast guanylyltransferases has revealed a common motif around a lysine residue at the amino-terminal part of the proteins [Y(V, A)X2K(T, A)DG] which resembles the adenylylation site of DNA ligases (Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991). Proc. Natl. Acad. Sci. USA 88, 400-404). This lysine residue could be the guanylylation site in these enzymes.