thi4 mutants of Schizosaccharomyces pombe exhibit defective thiamine biosynthesis, and thi4 mutations define a gene which is believed to be involved in the phosphorylation of 4-amino-5-hydroxymethyl-2-methylpyrimidine or 5-(2-hydroxyethyl)-4-methylthiazole and/or in the coupling of the two phosphorylated precursors to thiamine monophosphate (A. M. Schweingruber, J. Dlugonski, E. Edenharter, and M. E. Schweingruber, Curr. Genet. 19:249-254, 1991). The thi4 gene was cloned by functional complementation of a thi4 mutant and physically mapped on the left arm of chromosome I close to the genetic marker gln1. The thi4-carrying DNA fragment shows an open reading frame encoding a protein of 518 amino acids and a calculated molecular mass of 55.6 kDa. The appearance of thi4 mRNA is strongly repressed by thiamine and to a lesser extent by 5-(2-hydroxyethyl)-4-methylthiazole. thi4 mRNA production is under the control of the thi1 gene-encoded transcription factor and of the negative regulators encoded by genes tnr1, tnr2, and tnr3. thi4 is expressed and regulated in manners similar to those of other S. pombe genes involved in thiamine metabolism, including thi2, thi3, and pho4.