The thymidine kinase encoded by herpes simplex virus type 1 contains an amino acid sequence homologous to a consensus sequence related to the ATP-binding site in many proteins. We have used site-directed mutagenesis to investigate the importance of the five highly conserved amino acids within this segment. When any one of the three glycines was changed to valine the corresponding mutant enzyme was inactive. The mutation of lysine 63 to isoleucine destroyed the enzymatic activity. When threonine 64 was changed to alanine the mutant enzyme lost its activity. However, when this threonine was changed to serine the enzyme was still active but with different apparent Michaelis constants (Km) for thymidine and ATP. The wild-type thymidine kinase has apparent Km's of 0.5 and 20 microM for thymidine and ATP, respectively, while the mutant enzyme displayed Km's of 2.3 and 60 microM for thymidine and ATP. These results indicate that this homologous segment is essential for the function of the thymidine kinase and is involved in the substrate binding domain of the enzyme.