We have previously reported that vasopressin activates chloride channels, leading to depolarization of glomerular mesangial cells via both calcium-dependent and calcium-independent pathways (S.G. Kremer, W.V. Breuer, and K. L. Skorecki, J. Cell. Physiol. 138: 97-105, 1989). However, the calcium-independent pathways were not defined. Using fluorescent probes, we now demonstrate that activation of protein kinase C (PKC) results in cellular depolarization in the absence of a calcium signal. This depolarization is also mediated by an enhanced conductance to chloride. Downregulation of PKC partially attenuated but did not abolish the depolarization response to vasopressin. Depolarization persisted when, in addition, calcium responses were also abolished and prostaglandin production was eliminated, suggesting an additional pathway for depolarization. G protein activation by aluminum fluoride also resulted in cellular depolarization mediated by an enhanced conductance to chloride, which persisted when calcium and PKC-signaling pathways were eliminated. This suggests the presence of a calcium- and PKC-independent pathway for G protein-mediated chloride-dependent depolarization. These findings point to the presence of at least three separate signaling pathways available for the activation of mesangial cell chloride channels, i.e., calcium, PKC, and a G protein.