TCR engagement triggers phospholipase Cgamma1 activation through the Lck-ZAP70-linker of activated T cell adaptor protein pathway. This leads to generation of diacylglycerol (DAG) and mobilization of intracellular Ca(2+), both essential for TCR-dependent transcriptional responses. TCR ligation also elicits transient recruitment of DAG kinase alpha (DGKalpha) to the lymphocyte plasma membrane to phosphorylate DAG, facilitating termination of DAG-regulated signals. The precise mechanisms governing dynamic recruitment of DGKalpha to the membrane have not been fully elucidated, although Ca(2+) influx and tyrosine kinase activation were proposed to be required. We show that DGKalpha is tyrosine phosphorylated, and identify tyrosine 335 (Y335), at the hinge between the atypical C1 domains and the catalytic region, as essential for membrane localization. Generation of an Ab that recognizes phosphorylated Y335 demonstrates Lck-dependent phosphorylation of endogenous DGKalpha during TCR activation and shows that pY335DGKalpha is a minor pool located exclusively at the plasma membrane. Our results identify Y335 as a residue critical for DGKalpha function and suggest a mechanism by which Lck-dependent phosphorylation and Ca(2+) elevation regulate DGKalpha membrane localization. The concerted action of these two signals results in transient, receptor-regulated DGKalpha relocalization to the site at which it exerts its function as a negative modulator of DAG-dependent signals.