Abstract Thymidine kinase is an important enzyme in the pyrimidine nucleotide salvage pathway and catalyzes the formation of thymidylate from thymidine using ATP as a phosphate donor. The gene encoding thymidine kinase of the oral bacterium Streptococcus gordonii was cloned and the nucleptide sequence determined. The inferred amino acid sequence of thymidine kinase (191 amino acids) exhibited 43% identity with type H thymidine kinase from Escherichia coli. The S. gordonii thymidine kinase expressed in Escherichia coli KY895 (tdk−) was inhibited by thymidline triphosphate, a feature typical of type II thymidine kinases. Immediately 3′ to the tdk gene, and possibly co-transcribed with it, was the gene encoding release factor 1 (prfA).