Renal epithelia can be provoked to release nucleotides, which acutely increases [Ca2+ ]i via P2 receptors. Cultured cells often show spontaneous Ca2+ oscillations, a feature suggested to involve nucleotide signalling. Here, fluo-4 loaded MDCK cells are used as a model system to quantify and characterise [Ca2+ ]i increases in renal epithelia. The spontaneous [Ca2+ ]i increases occurred randomly as single cell events. During 1 minute 10.9±6.7% (n=23) of the cells oscillated with a frequency of 0.79±0.13 events/s in 300 cells. Scavenging apical ATP (apyrase, 10U/ml, hexokinase, 20U/ml) or inhibiting P2 receptors (suramin, 1mM) significantly reduced the spontaneous oscillatory activity. The ecto-ATPase inhibitor, ARL67156 (100mM), increased the [Ca2+ ]i oscillatory activity to 27.0±4.66 %/min (n=13). Spontaneous ATP release was directly detected from MDCK cells by the luciferin/luciferase reaction. The correlation between spontaneous [Ca2+ ]i oscillations and nucleotide signalling was further tested in 132 1N1 cells lacking P2 receptors. The cells hardly showed any spontaneous oscillations. However, after transfection with either hP2Y6 or hP2Y2 receptors the cells revealed a striking degree of oscillatory activity. Taken together these findings imply that renal epithelia spontaneously release nucleotides leading to P2-receptor activation. Thus, the purinergic signalling system is likely to regulate renal tubular function.