Treatment of human epithelial kidney (HEK293) cells with low concentrations of the muscarinic agonist methacholine results in the activation of complex and repetitive cycling of intracellular calcium ([Ca2+](i)), known as [Ca2+](i) oscillations. These oscillations occur with a frequency that depends on the concentration of methacholine, whereas the magnitude of the [Ca2+](i) spikes does not. The oscillations do not persist in the absence of extracellular Ca2+, leading to the conclusion that entry of Ca2+ across the plasma membrane plays a significant role in either their initiation or maintenance. However, treatment of cells with high concentrations of GdCl3, a condition which limits the flux of calcium ions across the plasma membrane in both directions, allows sustained [Ca2+], oscillations to occur. This suggests that the mechanisms that both initiate and regenerate [Ca2+](i) Oscillations are intrinsic to the intracellular milieu and do not require entry of extracellular Ca2+. This would additionally suggest that, under normal conditions, the role of calcium entry is to sustain [Ca2+](i) oscillations. By utilizing relatively specific pharmacological manoeuvres we provide evidence that the Ca2+ entry that supports Ca2+ oscillations occurs through the store-operated or capacitative calcium entry pathway. However, by artificial introduction of a non-store-operated pathway into the cells (TRPC3 channels), we find that other Ca2+ entry mechanisms can influence oscillation frequency in addition to the store-operated channels.