The stepwise synthesis of thymidine triphosphate (TTP) requires a kinase for phosphorylation in the last step. Because pyruvate kinase (PK) using phosphoenolpyruvate (PEP) as substrate can regenerate adenosine triphosphate and phosphorylate thymidine diphosphate as well, we chose this enzyme for the synthesis of TTP via an enzymatic cascade reaction. The metalloenzyme PK shows pronounced promiscuity and therefore fits well to the conditions of this reaction. PK commonly used today is isolated from rabbit muscle. We cloned and expressed the respective open reading frame in Escherichia coli, purified, and characterized the His-tagged recombinant enzyme. The enzyme has an activity optimum at 37°C and in the pH range from 7.4 to 7.8. K(M) constants conformed well with the isolated native enzyme for adenosine diphosphate (ADP) to 0.37±0.02 mM and for PEP to 0.07±0.01 mM. The recombinant enzyme shows the following range in its substrate specificity: ADP>dADP>dGDP>dCDP>thymidine diphosphate (TDP). It allows the phosphorylation of TDP to TTP in high yield (up to 95%). The metal ions Mg(2+) and K(+) are necessary for full enzymatic activity. The addition of transition metal ions such as Mn(2+), Cu(2+), Co(2+), and Ni(2+) reduces activity. Storage of the enzyme at -20°C retains full activity.