The involvement of phosphatidylcholine-specific phospholipase C (PC-PLC) and D (PC-PLD) in the regulation of the thapsigargin-induced Ca2+ increase was investigated. Pretreatment of human lymphocytes with the PC-PLC inhibitors D609 or U73122 enhanced the thapsigargin-induced Ca2+ influx. By contrast, no effect was observed in the presence of phospholipase D inhibitor butanol. Addition of exogenous PC-PLC but not PC-PLD to lymphocytes prestimulated with thapsigargin led to a decrease of intracellular Ca2+. In addition, thapsigargin was shown to release diacylglycerol (DAG) from cellular phosphatidylcholine pools. The thapsigargin-induced DAG formation was inhibited by U73122 and D609 but not by butanol. Moreover, no formation of the PC-PLD activity marker phosphatidylbutanol was detected. Thapsigargin-induced DAG formation was dependent on the Ca2+ entry, as it was abolished in the absence of extracellular Ca2+ or in the presence of Ni2+. Further investigations demonstrated that the inhibition of the cellular DAG target, protein kinase C (PKC), enhanced thapsigargin-induced Ca2+ increase, whereas direct PKC activation had an inhibitory effect. Taken together, our results reveal the involvement of PC-PLC in the regulation of the thapsigargin-induced Ca2+ increase and point to the existence of a physiologic feedback mechanism activated by Ca2+ influx and acting via consecutive activation of PC-PLC and PKC to limit the rise of intracellular Ca2+.