Extracellular ATP acting on P2Y2 receptors evokes a biphasic Ca2+ elevation in HaCaT keratinocytes. Whereas the initial rise reflects Ca2+ release from intracellular stores, the second, slowly decaying phase is mediated by Ca2+ influx through presumed store- and receptor-operated channels. Interestingly, membrane potential recordings indicated that the second phase of the Ca2+ response is accompanied by a pronounced hyperpolarization which results from the activation of an intermediate conductance Ca2+-activated K+ channel (IK1). Using ratiometric Ca2+ imaging (calcium green/fura red), we investigated whether the IK1-mediated hyperpolarization is essential for Ca2+ influx. In HaCaT cells treated with 1-EBIO for 24 h to eliminate hIK1 (cf. Koegel et al., JBC 278: 3323–3330, 2003), the second phase of the Ca2+ response to ATP was significantly diminished. Ca2+ influx was reduced in an almost identical fashion when extracellular K+ was elevated to 31 mM to "clamp" HaCaT cells at their mean RMP (-40 mV) during ATP application. The present experiments demonstrate that IK1 plays a crucial role in receptor-mediated Ca2+ signaling.