A complex phospholipid from bovine vitreous body with a strong Ca(2+)-mobilizing activity has been recently isolated to homogeneity by our group. In this work, a sequential analysis of its transmembrane signaling pathway has been undertaken to characterize the intracellular mechanisms responsible for the Ca(2+) rise. The results show that this phospholipid induces, in a dose-dependent manner (ED(50) of around 0.25 microgram/ml), a Ca(2+) mobilization from inositol 1,4,5-trisphosphate-insensitive intracellular stores, with no participation of extracellular Ca(2+). Upon repeated administration, it shows no signs of autologous desensitization, does not induce heterologous desensitization of the L-alpha-lysophosphatidic acid (LPA) receptor but is desensitized by the previous administration of LPA. The Ca(2+)-mobilizing activity requires a membrane protein, is blocked after preincubation of the cells with pertussis toxin and phorbol esters, as well as by U73122 (an inhibitor of phospholipases C/D), R59022 (a diacylglycerol kinase inhibitor), and D609 (which inhibits phosphatidylcholine-specific phospholipase C). Upon administration of this phospholipid, the intracellular levels of phosphatidic acid (PA) rise with a time course that parallels that of the Ca(2+) mobilization, suggesting that PA could be responsible for this Ca(2+) signal. Exposure to AACOCF(3) (a specific inhibitor of phospholipase A(2)) does not modify the Ca(2+) rise, ruling out the possibility that the PA generated could be further converted to LPA by the action of phospholipase A(2). Based on the experimental data obtained, a signaling pathway involving a phosphatidylcholine-specific phospholipase C coupled to diacylglycerol kinase is proposed. This compound may represent a new class of bioactive lipids with a putative role in the physiology of the vitreous body.