In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores and in some cell types store-operated influx occurs through Ca2+ release activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions where cytoplasmic Ca2+ concentration can freely fluctuate, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was strongly buffered at low levels. Hence Ca2+-calmodulin is not required for the activation of CRAC channels per se, but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than Ca2+ extrusion. Hence these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to inhibit further Ca2+ influx. This slow inactivation can be activated by relative brief Ca2+ influx (30-60 seconds), reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.