nmt1 in fission yeast is essential for thiamine biosynthesis and is regulated by the thi1 transcription factor. The thiamine-repressible nmt1 promoter is the most widely used promoter construct for gene expression studies in fission yeast. We show that in addition to thi1, thi5 also regulates the nmt1 promoter and its expression is undetectable in a thi1 thi5 double deletion. Thi5 over-expression relieves the repression of nmt1 by thiamine and rescues the thiamine auxotrophy in thi1 deletions. Thi5 may also work to regulate Thi1 activity. Sporulation defects and decreased conjugation were observed in a thi1 disruption; deleting thi5 did not affect conjugation, but resulted in decreased sensitivity to exogenous thiamine. The thi5 deletion is epistatic to thi1 with respect to the failure of the thi1 disruption to produce spores. Thi5 negatively regulates some stages of meiosis. Over-expressing Thi1 from its native promoter results in increased thiamine-insensitive conjugation in all genetic backgrounds, suggesting that Thi1 positively regulates meiosis and that thiamine inhibition of conjugation is a result of Thi1 repression. Thi5 and Thi1 work in the same pathway to positively regulate nmt1 promoter activity. In conjugation, Thi5 and Thi1 operate in different pathways to transcribe antagonists involved in the completion of meiosis.