Fungi possess efficient mechanisms of pH and ion homeostasis, allowing them to grow over a wide range of environmental conditions. In this study, we addressed the role of the pH response transcription factor PacC in salt tolerance of the vascular wilt pathogen Fusarium oxysporum. Loss-of-function pacC(+/-) mutants showed increased sensitivity to Li(+) and Na(+) and accumulated higher levels of these cations than the wild type. In contrast, strains expressing a dominant activating pacC(c) allele were more salt tolerant and had lower intracellular Li(+) and Na(+) concentrations. Although the kinetics of Li(+) influx were not altered by mutations in pacC, we found that Li(+) efflux at an alkaline, but not at an acidic, ambient pH was significantly reduced in pacC(+/-) loss-of-function mutants. To explore the presence of a PacC-dependent efflux mechanism in F. oxysporum, we cloned ena1 encoding an orthologue of the yeast P-type Na(+)-ATPase ENA1. Northern analysis revealed that efficient transcriptional activation of ena1 in F. oxysporum required the presence of high Na(+) concentrations and alkaline ambient pH and was dependent on PacC function. We propose a model in which PacC controls ion homeostasis in F. oxysporum at a high pH by activating expression of ena1 coordinately with a second Na(+)-responsive signaling pathway.