Thymidine phosphorylase (TP; EC 22.214.171.124) catalyzes the reversible phosphorolysis of thymidine, deoxyuridine, and their analogues to their respective nucleobases and 2-deoxy-α-D-ribose-1-phosphate (dRib-1-P). TP is a key enzyme in the pyrimidine salvage pathways. Activity of the enzyme is crucial in angiogenesis, cancer chemotherapy, radiotherapy, and tumor imaging, Nevertheless, a complete set of kinetic parameters has never been reported for any human TP. This study describes the kinetic mechanism and regulation of native human hepatic TP. The liver is a main site of pyrimidine metabolism and contains high levels of TP. Initial velocity and product inhibition studies demonstrated that the basic mechanism of this enzyme is a sequential random bi-bi mechanism. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation, and a binding pattern indicating that the binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were K Thymidine = 284 ± 55, K Pi = 5.8 ± 1.9, K Thymine = 244 ± 69, and K dRib-1-P = 90 ± 33 μM. Thymine was a product activator, but becomes a substrate inhibitor at concentrations eight times higher than its K m . dRib-1-P was a non-competitive product inhibitor of the forward reaction. It bounded better to the Enzyme●P i complex than the free enzyme, but had better affinity to the free enzyme than the Enzyme●Thymidine complex. In the reverse reaction, dRib-1-P enhanced the binding of thymine. The enhancement of the thymine binding along with the fact that dRib-1-P was a non-competitive product inhibitor suggests the presence of another binding site for dRib-1-P on the enzyme.