We evaluated the contribution of CaR-mediated G_i-coupled signaling to TNF production in mTAL cells. A selective G_i inhibitor, Pertussis toxin (PTX), but not the inactive B-oligomer binding subunit, abolished CaR-mediated increases in TNF production. The inhibitory effect of PTX was partially reversed by using an adenylate cyclase inhibitor. CaR-mediated TNF production also was partially reversed by a cAMP analog, 8-Br-cAMP. IP1 accumulation was CaR-dependent and blocked by PI-PLC; partial inhibition also was observed with PTX. CaR increased calcineurin (CaN) activity by approximately 3-fold and PTX prevented CaR-mediated increases in CaN activity, an NFAT cis-reporter construct, and a TNF promoter construct. The interaction between G_i and PKC was determined, as we previously showed that CaR-mediated TNF production was CaN- and NFAT-mediated and G_q-dependent. CaR activation increased PKC activity by 2-fold, an effect abolished by transient transfection with a dominant negative CaR construct, R796W, or pretreatment with PTX. Inhibition with the pan specific PKC inhibitor, GF 109203X (20 nM) abolished CaR-mediated increases in activity of CaN, an NFAT reporter, and a TNF promoter construct. Collectively, the data suggest that G_i-coupled signaling contributes to NFAT-mediated TNF production, in a CaN- and PKC-dependent manner, and may be part of a CaR mechanism to regulate mTAL function. Moreover, concurrent G_q and G_i signaling is required for CaR-mediated TNF production in mTAL cells via a CaN/NFAT pathway that is PKC-dependent. Understanding CaR-mediated signaling pathways that regulate TNF production in the mTAL is crucial to defining novel mechanisms that regulate extracellular fluid volume and salt balance.