sti35 is one of the heat-shock genes in Fusarium oxysporum, which is a fungal pathogen for wilt disease in plants. We have isolated a genomic clone of sti35 and used it to create disruption mutations. Disruption of the sti35 coding region resulted in the loss of a 32-kDa protein present in heat-shocked cells. The disruption had no detectable effect on growth and development at various temperatures, nor on the ability to acquire thermotolerance in nutrient medium. But the sti35 disruptants showed increased thermotolerance, relative to the wild-type strain, when incubated in minimal medium after heat treatment.