Treatment of Fusarium oxysporum with 5-azacytidine, a potent inhibitor of DNA methylation, induced nonpathogenic mutants. Analysis of the protein expression pattern by two-dimensional gel electrophoresis revealed a protein that is present in yeast-form cells of the mutants but absent in those of the wild-type strain. N-terminal amino acid analysis indicated that this protein is identical to a region of a polypeptide encoded by a cDNA clone, sti35, previously identified as a heat shock gene in F. oxysporum. A genomic clone for sti35 was isolated and sequence analysis revealed an intron and two heat shock elements upstream of sti35. The analysis also revealed the presence of a leader sequence composed of 27 amino acid residues, which shares a common amino acid composition with leader sequences of the proteins located in the mitochondrial matrix. Different expression patterns of sti35 in the mutants and wild-type strain were demonstrated.