Release of Ca2+ from intracellular pools is the main trigger for activation of Ca2+ entry during the generation of Ca2+ signals in non-excitable cells. The aim of these studies is to investigate the relationship between Ca2+ pool emptying and activation of Ca2+ influx. Using the smooth muscle cell line, DDT1MF-2, changes in cytosolic Ca2+ concentration were monitored by loading cells with the fluorescent Ca2+ indicator, fura-2. The studies reveal that Ca2+ pool depletion by treatment with the SERCA pump inhibitors, thapsigargin and 2,5-Di-tert-butylhydroquinone (DBHQ), stimulates the activation of a novel and distinct Ca2+ entry pathway sensitive to caffeine. Refilling of Ca2+ pools occurs concomitantly with the disappearance of the caffeine-sensitive Ca2+ entry pathway. Stimulation of the caffeine-sensitive Ca2+ entry pathway occurs independently of the means used to deplete intracellular Ca2+ pools (pump inhibition or ionophore-induced depletion). Therefore, activation of the caffeine-sensitive Ca2+ entry pathway is directly controlled by Ca2+ pool depletion. Ion permeability studies indicate that the Ca2+ entry pathway sensitive to caffeine is less selective for Ca2+ than store-operated Ca2+ entry, the major Ca2+ influx pathway activated by pool depletion in a wide variety of cells. Nitric oxide-induced thiol modification (nitrosylation) has recently been shown to have major modulatory effects on the activity of several Ca2+ channels. Therefore, it was important to investigate the role of thiol nitrosylation in controlling Ca2+ entry and its activation by pool depletion. Studies reveal that nitric oxide donors activate a substantial entry of Ca2+ through a direct pathway which is independent of guanylate cyclase activation, a well-studied target for nitric oxide. Cell permeant alkylating agents activate an entry of Ca2+ remarkably similar to nitric oxide-induced Ca2+ entry, indicating that activation of Ca2+ entry relies on modification of one or more thiol residues in the channel or a closely associated protein. Most significantly, Ca2+ pool emptying strongly stimulates the Ca2+ entry activated by both nitric oxide donors and alkylating agents, revealing a direct link between thiol nitrosylation and activation of a store-sensitive Ca2+ entry pathway. These studies provide further evidence for the strong involvement of Ca2+ pool content in controlling the activity of major Ca2+ entry mechanisms.