The mitogenic activity of endothelin and its ability to stimulate PtdIns(4,5)P2 and phosphatidylcholine turnover in Rat-1 fibroblasts was studied. Stimulated incorporation of [3H]thymidine occurred in the absence of any other added growth factors. The endothelins stimulated rapid generation of both Ins(1,4,5)P3 and choline. Endothelin-1 and endothelin-2 were equipotent in stimulating both responses, but endothelin-3 was less potent. Endothelin-1-stimulated Ins(1,4,5)P3 generation reached a maximum at 5 s and then declined; however, the response was long-lived, with a 4.5-fold elevation over basal still observed after 15 min. Endothelin-stimulated choline generation was observed with no increase in choline phosphate; indeed, the apparent level of this metabolite fell after 30 min of stimulation, presumably due to the observed stimulation of phosphatidylcholine synthesis. The endothelin-stimulated increase in choline generation was abolished in cells where protein kinase C was down-regulated. However, endothelin-stimulated choline generation was greater than that observed in response to a protein kinase C-activating phorbol ester, raising the possibility that the peptide activates phospholipase D by both protein kinase C-dependent and -independent mechanisms.