The inwardly rectifying anion channel ClC-2 contributes to the regulation of neuronal excitability, Cl^- secretion and cell volume. As shown previously, ClC-2 is stimulated by the serum- and glucocorticoid-inducible kinase SGK1, an effect partially accounted for by inhibition of the ubiquitin ligase Nedd4-2 and mimicked by the SGK1 isoforms SGK2 and SGK3 as well as by the related kinase PKB/Akt. SGK1 and PKB/Akt have been shown to phosphorylate and thus activate the kinase PIKfyve, which mediates some effects of SGK1 and PKB/Akt on cell membrane proteins. Employing the heterologous expression system of Xenopus oocytes, the present study explored whether PIKfyve participates in the regulation of ClC-2. Expression of wild type PIKfyve markedly increased the Cl^- conductance in oocytes injected with ClC-2 mRNA but not in oocytes injected without ClC-2 mRNA. The effect of PIKfyve coexpression on ClC-2 activity was mimicked by coexpression of constitutive active S422DSGK1. Coexpression of the SGK1 resistant S31APIKfyve mutant did not stimulate ClC-2 and blunted the stimulating effect of S422DSGK1. Conversely, coexpression of the inactive K127NSGK1 mutant did not stimulate ClC-2 and blunted the stimulating effect of wild type PIKfyve. In conclusion, SGK1 and PIKfyve concert to stimulate the Cl^- channel ClC-2.