THI4, a Saccharomyces cerevisiae gene originally identified as a result of transient expression in molasses medium and named MOL1 is regulated by thiamine. Using a THI4 promoter-lacZ fusion on a centromeric yeast vector, we have shown that the THI4 is completely repressed throughout batch culture by thiamine at a concentration around 1 microM, but shows high level constitutive expression in thiamine-free medium. The transient expression pattern observed in molasses medium can be mimicked by the addition of 0.15 microM-thiamine to defined minimal medium. Cells grown in thiamine-free medium have an intracellular thiamine concentration of around 9 pmol/10(7) cells. A low level (1 microM) of exogenous thiamine is completely sequestered from the medium within 30 min; intracellular thiamine concentrations rise rapidly, followed by a gradual decrease as a result of dilution during growth. A saturating extracellular level of thiamine leads to a maximal intracellular concentration of around 1600 pmol/10(7) cells, at which point the transport system is shut down. After transfer from repressing to non-repressing medium, THI4 becomes induced when the intracellular concentration of thiamine falls to 20 pmol/10(7) cells. A thi4::URA3 disruption strain is auxotrophic for thiamine, but can grow in the presence of hydroxyethyl thiazole, indicating that the gene product is involved in the biosynthetic pathway leading to the formation of the thiazole precursor of thiamine.