The diacylglycerol (DAG) signal generated from membrane phospholipids by hormone-activated phospholipases is attenuated by mechanisms that include lipolysis or phospholipid resynthesis. To determine whether the DAG signal might also be terminated by incorporation of DAG into triacylglycerol (TAG), we studied the direct formation of TAG from endogenous DAG generated by bacterial phospholipase C (PLC). When Chinese hamster ovary (CHO) cells prelabeled with [¹⁴C]oleate were treated with PLC from <i>Clostridium perfringens</i> for 6 h, [¹⁴C]phospholipid decreased 15% and labeled TAG increased 60%. This transfer of ¹⁴C label was even greater when the cells were simultaneously exposed to PLC and 100 μM oleic acid. PLC as well as oleate treatment concomitantly increased the TAG mass within the cell. Moreover, when phospholipids were prelabeled with [³H]glycerol, a subsequent increase in [³H]TAG indicated that an intact DAG moiety was channeled into the TAG structure. Incubating CHO cells with the diacylglycerol kinase inhibitor R59022 enhanced the formation of TAG from phospholipids hydrolyzed by PLC or by PLC in the presence of 100 μM oleate, but not by incubation with oleate alone, indicating that the DAG released from plasma membrane phospholipids does not require the formation of a phosphatidic acid precursor for TAG synthesis. Similarly, the diacylglycerol lipase inhibitor RHC 80267 did not alter TAG synthesis from plasma membrane DAG, further supporting direct incorporation of DAG into TAG. These studies indicate that DAG derived from plasma membrane phospholipid is largely used for TAG formation, and support the view that this mechanism can terminate DAG signals. The studies also suggest that a transport mechanism exists to move plasma membrane-derived DAG to the endoplasmic reticulum.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plata