Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca2+ influx. While Ca2+ influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca2+ release-activated Ca2+ (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca2+ release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca2+ influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca2+ store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.