Abstract: Genes encoding thiamine biosynthesis enzymes in microorganisms are tightly regulated, such that low environmental thiamine concentrations activate transcription and high concentrations are repressive. We have determined that multiple thiamine (THI) genes in Saccharomyces cerevisiae are also regulated by the intracellular NAD(+) concentration via the NAD(+)-dependent histone deacetylase Hst1, and to a lesser extent Sir2. Both of these HDACs associate with a distal region of the affected THI gene promoters that does not overlap with a previously defined enhancer region bound by the thiamine-responsive Thi2/Thi3/Pdc2 transcriptional activators. The specificity of histone H3 and/or H4 deacetylation carried out by Hst1 and Sir2 at the distal promoter region depends on the THI gene being tested. Hst1/Sir2-mediated repression of the THI genes occurs at the level of basal expression, thus representing the first set of transcription factors shown to actively repress this gene class. Importantly, lowering the NAD(+) concentration and inhibiting the Hst1/Sum1 HDAC complex elevated the intracellular thiamine concentration due to increased thiamine biosynthesis and transport, implicating NAD(+) in the control of thiamine homeostasis.