The plasma membrane Ca2+-ATPase (PMCA) plays a key role in the regulation of the intracellular Ca2+ concentration. Ethanol stimulates this Ca2+ pump in an isoform-specific manner. On search for a physiological molecule that could mimic the effect of ethanol, we have previously demonstrated that some sphingolipids containing free ‘‘hydroxyl” groups, like ceramide, are able to stimulate the PMCA. Since diacylglycerol (DAG) structurally shares some characteristics with ceramide, we evaluate its effect on the PMCA. We demonstrated that DAG is a potent stimulator of this enzyme. The activation induced is additive to that produced by calmodulin, protein-kinase C and ethanol, which implies that DAG interacts with the PMCA through a different mechanism. Additionally, by different fluorescent approaches, we demonstrated a direct binding between PMCA and DAG. The results obtained in this work strongly suggest that DAG is a novel effector of the PMCA, acting by a direct interaction.