Thymidine kinase (TK1) is a key enzyme in the salvage pathway of deoxyribonucleotide metabolism catalyzing the first step in the synthesis of dTTP by the transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5´-hydroxyl group of thymidine forming dTMP. Human TK1 is cytosolic and its activity is absent in resting cells, appears in late G1, increases in S phase coinciding with the increase in DNA synthesis and disappears during mitosis. The fluctuation of TK1 through cell cycle is important for providing a balanced supply of dTTP for DNA replication and is partly due to regulation of TK1 expression at the transcriptional level. However, TK1 is also a regulatory enzyme that can interchange between its dimeric and tetrameric forms, which have low and high catalytic efficiencies, respectively, depending on pre-assay incubation with ATP. Here, it is investigated which part of ATP is necessary for tetramerisation and how the reaction velocity is influenced by the enzyme concentration. The results show that only two or three of the phosphate groups of ATP are necessary for tetramerisation, and that kinetics and tetramerisation are closely related. Furthermore, enzyme concentration was found to have a pivotal effect on catalytic efficiency.