This study aims to investigate purinergic P2X and P2Y receptors in Ca²⁺ homeostasis in freshly isolated monolayers of murine retinal pigment epithelium (RPE) compared with cells of human ARPE-19 cell line. RPE cells were isolated from murine eyes under Ca²⁺-free conditions and held over night prior Ca²⁺ measurement. Intracellular free calcium ([Ca²⁺]i) was monitored using the Fura-2 Ca²⁺ imaging method. In ARPE-19 cells activation of purinergic receptors with ATP (100nM) led to a triphasic change in [Ca²⁺]i: an initial fast peak increase was followed by a slight transient decrease below resting [Ca²⁺]i from which a sustained increase followed. Emptying intracellular Ca²⁺ stores by Thapsigargin (1mM) prior to ATP application resulted in an ATP induced Ca²⁺ response with reduced initial peak. Freshly isolated RPE cells showed a heterogeneous pattern of ATP responses caused by a possible functional differentiation. In those cells showing a comparable ATP response only an initial peak and a very slow increase in [Ca²⁺]i could be seen under enduring ATP application. This leads us to the conclusion that RPE cells which contain melanosomes show different ATP responses due to different Ca²⁺-buffer capacitance.