Cells extracts from Thermus thermophilus HB27 express phosphorolytic activities on purines and pyrimidine nucleosides. Five putative encoding genes were cloned and expressed in Escherichia coli, and the corresponding recombinant proteins were purified and studied. Two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides in vitro, and the three were named TtPNPI, TtPNPII, and TtPyNP, respectively. The optimal temperature for the activity of the three enzymes was beyond the water boiling point and could not be measured accurately, whereas all of them exhibited a wide plateau of optimal pHs that ranged from 5.0 to 7.0. Analytical ultracentrifugation experiments revealed that TtPNPI was a homohexamer, TtPNPII was a monomer, and TtPyNP was a homodimer. Kinetic constants were determined for the phosphorolysis of the natural substrates of each enzyme. Reaction tests with nucleoside analogues revealed critical positions in the nucleoside for its recognition. Activities with synthetic nucleobase analogues, such as 5-iodouracil or 2,6-diaminopurine, and arabinosides were detected, supporting that these enzymes could be applied for the synthesis of new nucleoside analogs with pharmacological activities.