The amino acid sequence of 14 thymidine kinases and three other nucleotide binding enzymes have been compared by alignment of their primary and secondary structure. The overall alignment revealed five homologous regions, which are supposed to be part of the active site with a common three dimensional structure. Analysis of mutant enzymes brings further evidence for the importance of those regions. Single point mutations are responsible for an amino acid exchange within the homologous sequences thereby affecting the normal function of the enzymes. The substituted amino acids are essential for the binding function and, therefore, building part of an active site. After identification of the homologous regions we tried to fit the HSV 1 thymidine kinase on the known 3D-structure of adenylate kinase to reconstruct the essential binding regions of thymidine kinase as far as possible.