Abstract Among the orthopoxviruses variola virus induces in cells a characteristic thymidine kinase (TK) activity that can be feedback inhibited in reactions with thymidine triphosphate. Northern blot analyses of variola and monkeypox virus-infected cell extracts showed RNAs of the same molecular weight as the major (590-base) and minor (2380-base) TK transcripts described for vaccinia virus. The nucleotide sequences of 1275 bp in the TK gene region of variola and monkeypox viruses have been determined. When these sequences were compared with such sequences reported for vaccinia virus, differences were observed at 41 nucleotide positions. Examination of the putative encoded TK polypeptide for the three viruses revealed variation at eight amino acid positions. Two major differences in the amino acid composition of the variola virus TK were identified that might play a role in alteration of its kinetic properties.