We have shown that the plant pathogen Alternaria brassicicola exhibited very high susceptibility to ambruticin VS4 and to a lesser extent to the phenylpyrrole fungicide fludioxonil. These compounds are both derived from natural bacterial metabolites with antifungal properties and are thought to exert their toxicity by interfering with osmoregulation in filamentous fungi. Disruption of the osmosensor group III histidine kinase gene AbNIK1 (for A. brassicola NIK1) resulted in high levels of resistance to ambruticin and fludioxonil, while a mutant isolate characterized by a single-amino-acid substitution in the HAMP domain of the kinase only exhibited moderate resistance. Moreover, the natural resistance of Saccharomyces cerevisiae to these antifungal molecules switched to sensitivity in strains expressing AbNIK1p. We also showed that exposure to fludioxonil and ambruticin resulted in abnormal phosphorylation of a Hog1-like mitogen-activated protein kinase ( MAPK) in A. brassicicola. Parallel experiments carried out with wild-type and mutant isolates of Neurospora crassa revealed that, in this species, ambruticin susceptibility was dependent on the OS1-RRG1 branch of the phosphorelay pathway downstream of the OS2 MAPK cascade but independent of the yeast Skn7-like response regulator RRG2. These results show that the ability to synthesize a functional group III histidine kinase is a prerequisite for the expression of ambruticin and phenylpyrrole susceptibility in A. brassicicola and N. crassa and that, at least in the latter species, improper activation of the high-osmolarity glycerol-related pathway could explain their fungicidal properties