The Neurospora crassa homologue of the yeast no message in thiamine ( nmt-1) gene was characterized. The deduced 342-amino-acid gene product has more than 60% identity with other fungal homologues and 42% similarity to a putative bacterial permease. In addition to three introns disrupting the coding sequence, a differentially spliced intron in the 5' untranslated region was also detected. Unlike other fungi, the N. crassa nmt-1 gene is repressed only 6- to 8-fold by exogenous thiamine concentrations above 0.5 microM; and a high basal level of nmt-1 mRNA persists even at 5 microM thiamine. Immuno-blotting with purified antibodies detected two variants of NMT-1 which differ in size and charge. The more abundant 39-kDa form is more strongly repressed by thiamine than the 37-kDa protein. NMT-1 abundance modulates slowly in response to changes in the concentration of exogenous thiamine, suggesting that N. crassa maintains thiamine reserves in excess of immediate needs. Disruption of the nmt-1 gene demonstrated that it is essential for growth in the absence of exogenous thiamine. NMT-1-deficient strains had a growth rate and colony density which was about 70% of the wild type, despite supplementation with a wide range of exogenous thiamine. These results suggest that the nmt-1 gene plays some other role in addition to thiamine biosynthesis.