Exogenous thiamine regulates Aspergillus oryzae thiA, which is involved in thiamine synthesis. One of the two introns in its 5'-untranslated region (5'-UTR) contains motifs (regions A and B) highly conserved among fungal thiamine biosynthesis genes. Deletion of either region relieved the repression by thiamine and thiamine inhibited intron splicing, suggesting that regions A and B are required for efficient splicing. Furthermore, transcript splicing was essential for thiA gene expression. These observations suggest a novel gene expression regulatory mechanism in filamentous fungi, in which exogenous thiamine controls intron splicing to regulate gene expression. Interestingly, regions A and B constitute a part of a thiamine pyrophosphate-binding riboswitch-like domain that has been quite recently found in the 5'-UTR of thiA.