Agonist-induced cytoplasmic calcium signals often have profound effects on the membrane potential during cellular activation. In the present study, we report that cytoplasmic calcium elevation can regulate the membrane potential by a novel mechanism. TRESK, a recently described member of the two-pore domain potassium (2PK(+)) channel family, was activated 5-15-fold after stimulation of various Ca(2+)-mobilizing receptors in Xenopus oocytes. Extracellular application of ionomycin, as well as the microinjection of inositol 1,4,5-trisphosphate or calcium, also evoked TRESK activation, whereas microinjection of EGTA or pretreatment of the oocytes with thapsigargin prevented the receptor-mediated effect. These data indicate that TRESK is activated by increased cytoplasmic calcium concentration. However, application of Ca(2+) to inside-out membrane patches failed to influence TRESK single channel activity, suggesting that cytoplasmic factors are also required for the regulation. Cyclosporin A and FK506, specific inhibitors of the calcium/calmodulin-dependent protein phosphatase (calcineurin), completely eliminated TRESK activation. Coexpression of a constitutively active form of calcineurin with TRESK increased the basal background K(+) current and attenuated the response of the channel to the calcium signal, indicating that TRESK was activated by the permanent calcineurin activity. Serine 276 was identified as the major functional target of calcineurin in TRESK by alanine-scanning mutagenesis. This is the first example of calcineurin being involved in the regulation of a two-pore domain K(+) channel, and thus, TRESK channels may regulate the excitability of neurons and other cell types in response to Ca(2+)-mobilizing hormones and neurotransmitters in a manner that is sensitive to immunosuppressive drugs.