The sti35 gene of the vascular wilt fungus Fusarium oxysporum was originally identified based on induced expression under stress conditions. In this study, the transcriptional regulation and biological function of sti35 were examined in the tomato pathogen F. oxysporum f.sp. lycopersici. Expression of sti35 was repressed by thiamine and induced by high temperatures. Sti35 transcripts were detected both during early and late stages of infection of tomato plants by F. oxysporum. Heterologous expression of the sti35 cDNA restored thiamine prototrophy in a Saccharomyces cerevisiae thi4 mutant and increased UV tolerance in a uvr(-) mutant of Escherichia coli. Targeted Deltasti35 knockout mutants of F. oxysporum exhibited a thiamine auxotrophic phenotype and reduced tolerance to the superoxide-generating agent menadione, indicating that Sti35 has a dual role in thiamine biosynthesis and oxidative stress response. RT-PCR analysis revealed the presence of differential RNA splicing of the second 5'-UTR intron, suggesting that thiamine may regulate sti35 expression via a post-transcriptional mechanism. F. oxysporum transformants carrying a transcriptional fusion of the sti35 promoter to the lacZ reporter gene produced high levels of beta-galactosidase activity when grown in the absence, but not in the presence of thiamine. Thus, the sti35 promoter represents a useful tool for the controlled expression of genes of interest in F. oxysporum.