Effective fluid secretion in parotid acinar cells is dependent on the spatio-temporal regulation of intracellular Ca²⁺ ([Ca²⁺]_i) signals by cAMP. Such signalling cross-talk allows exquisite control of spatially distinct ion fluxes which maintain maximum water movement. Previous studies have demonstrated that the key molecular mechanisms for this signalling cross-talk is the PKA-mediated modulation of Ca²⁺ release and Ca²⁺ clearance [1,2]. The plasma membrane Ca²⁺-ATPase (PMCA), an important Ca²⁺ clearance pathway, maintains low resting [Ca²⁺]_i and is also dynamically regulated by Ca²⁺ in an integrative manner, important for modulating [Ca²⁺]_i oscillations. In addition, the PMCA binds to multi-protein signalling complexes, important for regulating local [Ca²⁺]_i signals and thus local Ca²⁺-dependent effectors. Previous studies have demonstrated in parotid acinar cells that PKA potentiates and phosphorylates the PMCA but only in the presence of [Ca²⁺]_i-raising agents (2). Therefore, the aim of the present study was to determine, (i) the expression and spatial distribution of specific PMCA isoforms; (ii) which of these PMCA isoforms are regulated by PKA in a Ca²⁺-dependent manner and, (iii) how this differentially regulates the spatial [Ca²⁺]_i clearance in parotid acinar cells. Fura-2-loaded parotid acinar cells were treated with 30 µM cyclopiazonic acid to inhibit the ER Ca²⁺-ATPase (SERCA) and raise [Ca²⁺]_i. Clearance of [Ca²⁺]_i was initiated by removal of external Ca²⁺ and normalised by fitting to an exponential decay to obtain a time constant (τ). Activation of PKA (using 10 µM forskolin) differentially potentiated [Ca²⁺]_i clearance in the apical region (τ=32.2 ± 1.3), compared to the basal region (τ=56.2 ± 1.0; n=5, p<0.05 using a non-parametric Mann-Whitney test), whereas in control cells apical and basal [Ca²⁺]_i clearance was not significantly different (apical τ=49.2 ± 2.8, basal τ=48.4 ± 2.0; n=4). Western blotting revealed that PMCA1, 2 and 4 are expressed in parotid acinar cells (n≥4). Immunofluorescence revealed that PMCA1 (n=3) was distributed throughout all regions of the plasma membrane, whereas PMCA4 (n=3) was localized to the apical membrane of parotid acinar cells. Likewise, the PDZ-containing accessory proteins, ezrin and EBP50 also exhibited an apical distribution. In situ phosphorylation assays demonstrated that PMCA1 (n=3) and PMCA2 (n=3) were phosphorylated by the combined treatment with forskolin and CCh. Collectively these data suggest that PMCA1 (or PMCA2) is phosphorylated by PKA in a Ca²⁺-dependent manner that differentially regulates Ca²⁺ clearance in the apical region of parotid acinar cells. This probably involves a Ca²⁺-mediated assembly of a signalling complex that brings PKA closer to the PMCA allowing targeted regulation specifically at the apical plasma membrane. Such tight spatial regulation of Ca²⁺ efflux may represent an important mechanism for the fine-tuning of Ca²⁺-dependent effectors at the apical membrane important for the regulation of fluid secretion and exocytosis.