Phospholipid signalling mediated by endothelin (ET) receptor subtypes was studied in the rat proximal tubule. In freshly isolated proximal tubule cells, ET-1, ET-2 and sarafotoxin S6c (S6c) evoked an increase in 1,2-diacylglycerol (DAG), inositol 1,4,5-trisphosphate (InsP3) and phosphocholine (PCho), suggesting stimulation of both phosphatidyl-inositol 4,5-bisphosphate- and phosphatidyl-choline-specific phospholipase C (PLC), while ET-3 increased only DAG and PCho, presumably via phosphatidyl-choline-dependent PLC. Renal cortical slices were also stimulated by the above-mentioned agonists, followed by isolation of either brush border (BBM) or basolateral (BLM) membranes for which mass measurements of inositol lipids and DAG were performed. In BBM, DAG increased in response to ET-1, ET-2 and ET-3, and was followed by protein kinase C (PKC) translocation to the BBM, while in BLM, DAG formation and translocation of PKC were observed only in response to ET-3, suggesting spatial segregation of signalling systems between two membane domains of proximal tubule cells. Tyrphostine, pertussis toxin (PTX) or cholera toxin (CTX) did not influence ET-mediated signalling in either of the membranes, suggesting involvement of PTX- and CTX-insensitive G-protein-mediated stimulation of PLCbeta by ET receptors. ET-dependent stimulation of PLC in BBM and BLM was used as a tool to examine the presence of different ET receptor subtypes in these two cell membrane domains. BQ123, an inhibitor of ETA receptors, did not prevent ET-1-mediated signalling in BBM, but an ETA,B antagonist, bosentan, inhibited ET-3-mediated signalling in BBM. In addition, an ETB agonist, S6c, stimulated PLC in BBM. Neither BQ123 nor bosentan inhibited ET-3 signalling in BLM. Therefore, these data strongly suggest the presence of ETB receptors coupled to phosphatidyl-inositol 4,5-bisphosphate- and phosphatidyl-choline-dependent PLC in BBM and ETC receptors linked to phosphatidyl-choline-dependent PLC in BLM.