The cDNA clone encoding human thymidine kinase (hTK), was expressed in E. coli using a prokaryotic expression vector, pKK 223-3. The kinetics of the recombinant hTK (rhTK) were similar to those of cytosolic TK but not of mitochondrial TK. rhTK was highly purified in the presence of either ATP or dithiothreitol (DTT). The specific activity of rhTK purified in the presence of ATP [rhTK(ATP)] was lower than that of rhTK purified in the presence of DTT [rhTK(DTT)]. Activity of the purified rhTK(ATP) was enhanced by addition of thiols including DTT, cysteine, homocysteine and beta-mercaptoethanol but inhibited by various sulfhydryl reagents such as 5,5'-dithio-bis(2-nitrobenzoic acid). Hence, it was suggested that rhTK is a thiol-type enzyme. Apparent Mr of purified rhTK(ATP) was 100 kDa, which corresponds to the size of a tetramer (25 kDa subunit), while that of purified rhTK(DTT) was 50 kDa, the size of a dimer. The tetramer form of rhTK(ATP) was converted to the dimer by replacement of ATP by DTT. On the other hand, the dimer form of rhTK(DTT) was converted to the tetramer by addition of ATP. Thus, the catalytic activity of human cytosolic TK might be regulated by thiols as well as ATP via its polymerization status.