The serine kinase protein kinase D (PKD) has a cysteine-rich domain (CRD) that binds diacylglycerol (DAG) with high affinity. PKD is cytosolic in unstimulated T cells, but it rapidly polarizes to the immunological synapse in response to antigen/antigen presenting cells (APCs). PKD repositioning is determined by the accumulation of DAG at the immunological synapse and changes in DAG accessibility of the PKD-CRD. Unstimulated T cells are shown to have a uniform distribution of DAG at the plasma membrane, whereas after T cell activation, a gradient of DAG is created with a persistent focus of DAG at the center of the synapse. PKD is only transiently associated with the immune synapse, indicating a fine tuning of PKD responsiveness to DAG by additional regulatory mechanisms. These results reveal the immune synapse as a focal point for DAG and PKD as an immediate and dynamic DAG effector during T cell activation.