Herpes Simplex Virus type 1 thymidine kinase (HSV 1 TK) is a key target for antiviral therapy and it phosphorylates a broad spectrum of nucleosides and nucleotides. We report the results from kinetic and inhibition experiments with HSV 1 TK, and show that there is a preferred, but not exclusive, binding order of substrates, i.e. dT binds prior to ATP. Furthermore, the results provide new informations on the mechanism of binding suggesting that HSV1 TK undergoes conformational changes during the catalytic cycle.