Bovine adrenal glomerulosa cells are primed by an initial treatment with angiotensin II (AngII) to respond with enhanced secretion to a second exposure to AngII or to agents that increase calcium influx. We have hypothesized that the mechanism of priming involves a persistent increase in diacylglycerol (DAG) generated via sustained activity of phospholipase D (PLD). In this report, we sought to define the time frame of this priming response as well as to determine its mechanism using assays of aldosterone secretion, PLD activation and radiolabeled diacylglycerol levels. We found that in primary cultures priming was observed for up to 50 min following AngII washout, suggesting that the priming window is protracted in these cultures relative to freshly isolated cells. The phorbol ester, phorbol 12,13-dibutyrate (PDBu), was used to investigate the role of sustained PLD activation in the persistent DAG and priming responses. PDBu was able both to prime glomerulosa cells to respond with enhanced secretion to AngII and to elicit a persistent increase in DAG following PDBu washout. This persistent increase in DAG levels with an initial exposure to PDBu or AngII was not the result of maintained PLD activity after agent removal, since PLD activation returned to basal levels by 30 min after washout. Finally, inhibition of PLD signaling during the initial AngII treatment inhibited the subsequent response to AngII or to another agent that increases calcium influx. Thus, our results suggest that persistent DAG resulting from PLD signaling mediates the priming response to AngII or PDBu.