The purinergic receptors of GFSHR-17 granulosa cells were characterised by perforated patch-clamp and [Ca²⁺]_i imaging. We observed a resting intracellular Ca²⁺-concentration ([Ca²⁺]_i) of 100 nM and a membrane potential of -40 mV. This was consistent with high K⁺- and Cl^--permeability and a high intracellular Cl^--concentration of 40 mM. Application of ATP (10–50 mM) for 5–10 s, every three minutes stimulated repetitive depolarisation from resting potential of -40 mV to -30 mV followed by increase in [Ca²⁺]_i and hyperpolarisation to -70 mV. The Ca²⁺ and electrical responses to ATP were not altered by removal of external Ca²⁺ and were also elicited by UTP indicating expression of P2Y receptor family. Prolongation of application time to 20 s reduced the probability of repetitive stimulation, indicating a desensitisation of the receptors. Western blot and pharmacological dissection using UDP as well as the blockers of phospholipase C (U73122) or IP₃ receptor (2-APB) revealed that the response to ATP was related to P2Y₂ and P2Y₄ subtypes. Furthermore, blockage of Ca²⁺ release with U73122 or 2-APB suppressed hyperpolarisation and CPA, the blocker of SERCA pump induced [Ca²⁺]_i-increase followed by hyperpolarisation in absence of ATP indicating that [Ca²⁺]_i-increase was a prerequisite for the hyperpolarisation. However, the inhibitors of Ca²⁺-activated K⁺ channels apamin, clotrimazole or iberiotoxin did not alter the ATP related hyperpolarisation. Blockage of Cl^- channels hyperpolarised the cells to -70 mV and suppressed further ATP induced hyperpolarisation predicted by Goldmann-Hodgkin equation, under assumption that ATP were activating K⁺-channels. The results suggest that by regulating [Ca²⁺]_i, P2Y₂ and P2Y₄ receptors of the rat GFSHR-17 granulosa cells modulate Cl^- permeability and thereby the membrane potential.