The rat DRG CaR was stably expressed in-frame as an EGFP fusion protein in HEK293 cells, and is functionally linked to changes in [Ca²⁺]_i. RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA, and Western blot analysis of membrane proteins showed a doublet of 168-175 kDa and 185 kDa consistent with native and glycosylated forms of the CaR.EGFP fusion protein, respectively. Increasing [Ca²⁺]_e from 0.5 mM to 1 mM resulted in increases in [Ca²⁺]_i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca²⁺]_e-response studies indicate a Ca²⁺ sensitivity with an EC₅₀ of 1.75 ± 0.09 mM. NPS R-467 and Gd³⁺ activated the SN CaR-EGFP. When [Ca²⁺]_e was successively raised from 0.25 mM to 4 mM, peak [Ca²⁺]_i, attained with 0.5 mM, was reduced by approximately 50%. Similar reductions were observed with repeated applications of 10 mM Ca²⁺, 1 and 10 µM NPS R-467 or 50 and 100 µM Gd³⁺, indicating desensitization of the response. Furthermore, Ca²⁺ mobilization increased phosphorylated PKC levels in the cells. However, the PKC activator, phorbol myristate acetate (PMA) did not inhibit CaR-mediated Ca²⁺ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca²⁺_e. Treatment of cells with 100 nM PMA for 24 hr, to down-regulate PKC, reduced [Ca²⁺]_i transients by 49.9 ± 5.2% (at 1 mM Ca²⁺) and 40.5 ± 6.5% (at 2 mM Ca²⁺), compared to controls. The findings suggest involvement of PKC in the pathway for Ca²⁺ mobilization following CaR activation.