Fungal histidine kinases (HKs) have been implicated in different processes, such as the osmostress response, hyphal development, sensitivity to fungicides and virulence. Members of HK class III are known to signal through the HOG mitogen-activated protein kinase (MAPK), but possible interactions with other MAPKs have not been explored. In this study, we have characterized fhk1, encoding a putative class III HK from the soil-borne vascular wilt pathogen Fusarium oxysporum. Inactivation of fhk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides, as well as increased sensitivity to hyperosmotic stress and menadione-induced oxidative stress. The osmosensitivity of Delta fhk1 mutants was associated with a striking and previously unreported change in colony morphology. The Delta fhk1 strains showed a significant decrease in virulence on tomato plants. Epistatic analysis between Fhk1 and the Fmk1 MAPK cascade indicated that Fhk1 does not function upstream of Fmk1, but that the two pathways may interact to control the response to menadione-induced oxidative stress.