The thi80 mutant of Saccharomyces cerevisiae (Nishimura, H., Kawasaki, Y., Nosaka, K., Kaneko, Y., and Iwashima, A. (1991) J. Bacteriol. 173, 2716-2719) shows markedly reduced activity of thiamin pyrophosphokinase (TPK; EC 2.7.6.2). We have isolated a DNA fragment carrying the THI80 gene from a yeast genomic library by its ability to complement constitutive synthesis of the thiamin-repressible acid phosphatase, encoded by the PHO3 gene, of thi80 mutant cells. On the other hand, the thi80 locus was found to be located 3.3 centimorgans proximal to the smp3 locus on the right arm of chromosome XV by genetic mapping analysis, and one more fragment bearing the THI80 gene trailing SMP3 gene was obtained by the plasmid eviction method. The nucleotide sequence of the overlapped region between the two isolated DNAs contained an open reading frame of 957 base pairs, encoding a 319-amino acid polypeptide with a calculated molecular weight of 36,616. When the intact THI80 open reading frame was expressed as a fusion protein carrying three vector-encoded amino acids at its N terminus in Escherichia coli lacking TPK, marked TPK activity was detected in the procaryotic cells, proving that the THI80 gene of S. cerevisiae encodes a structural gene of TPK. A gene disruption experiment demonstrated that the THI80 gene was essential for growth, and therefore, revealed that TPK is the only enzyme capable of synthesizing thiamin pyrophosphate in yeast. Studies of Northern blot analysis and the enzyme assay demonstrated that the THI80 gene expression is regulated mainly at the mRNA level by the intracellular thiamin pyrophosphate and requires the positive regulatory factors encoded by THI2 and THI3 genes. However, unlike thiamin-repressible acid phosphatase and the enzymes involved in thiamin synthesis of S. cerevisiae, TPK was found to be expressed constitutively at a low level and incompletely repressed by exogenous thiamin.