Human cytosolic thymidine kinase (hTK1) is the key enzyme of the pyrimidine salvage pathway and phosphorylates thymidine to thymidine monophosphate, a precursor building block of the DNA. Wild-type hTK1 (hTK1W) as well as a truncated form of the enzyme (hTK1M) carrying deletions at the N- and C-terminal regions were cloned as His₆-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (∼35 mg) and truncated hTK1 (∼23 mg) per liter of culture. The protein was purified to near homogeneity. The chaperone DnaK was identified to be the major contaminant that could be removed by applying an additional ATP–MgCl₂ incubation and washing step. hTK1W was a permanent tetramer in solution, whereas the truncated construct hTK1M appears to be a dimer in absence and presence of substrates. Both hTK1W and hTK1M exhibit pronounced thermal stability with transition temperatures (T_m) of 71.7 and 73.4 °C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (ΔT_m ranging from 5.6 to 12.5 °C) and hTK1M (ΔT_m ranging from 0.8 to 5.3 °C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a K_M of 0.51 μM and a k_cat of 0.28 s⁻¹ for wild-type hTK1. The truncated hTK1M has a K_M of 0.87 μM and k_cat of 1.65 s⁻¹, thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.