The activity of persistent Ca(2)(+) sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca(2)(+) entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca(2)(+) sparklets by PKC-alpha and c-Src, both of which increase whole cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca(2)(+) sparklet activity? 2) Does PKC-alpha activate c-Src to produce persistent Ca(2)(+) sparklets? With the use of total internal reflection fluorescence microscopy, Ca(2)(+) sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Cav1.2c (the neuronal isoform of Cav1.2) constructs +/- active or inactive PKC-alpha/c-Src. Cells expressing Cav1.2c exhibited both low-activity and persistent Ca(2)(+) sparklets. Persistent Ca(2)(+) sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Cav1.2c constructs mutated at one of two COOH-terminal residues (Y(2)(1)(2)(2)F and Y(2)(1)(3)(9)F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y(2)(1)(2)(2)F but not Y(2)(1)(3)(9)F Cav1.2c abrogated the potentiating effect of c-Src on Ca(2)(+) sparklet activity. We could not detect a significant change in persistent Ca(2)(+) sparklet activity or density in cells coexpressing Cav1.2c + PKC-alpha, regardless of whether WT or Y(2)(1)(2)(2)F Cav1.2c was used, or after PP2 application, suggesting that PKC-alpha does not act upstream of c-Src to produce persistent Ca(2)(+) sparklets. However, our results indicate that persistent Ca(2)(+) sparklet activity is promoted by the action of c-Src on residue Y(2)(1)(2)(2) of the Cav1.2c COOH terminus.