Activation of Jurkat T cells with phytohemagglutinin (PHA), CD3 or CD2 monoclonal antibodies (mAbs) results in a marked inhibition of phosphatidylserine (PS) synthesis. Activation of Jurkat T cells with PHA in a Ca(2+)-free medium resulted in an arrest of PS synthesis which was not reversed by the addition of Ca2+. The use of BAPTA to chelate Ca2+ ions released from intracellular stores prevented PHA-induced inhibition of PS synthesis. In addition, it was found that during activation, in the presence of BAPTA, a net Ca2+ influx paralleled an increase in PS synthesis, demonstrating that Ca2+ uptake caused an enhanced PS synthesis rather than an inhibition. The use of a CD2 mAb, D66, able to mobilize exclusively Ca2+ from intracellular stores, resulted in 51% inhibition of PS synthesis. N-Ethylmaleimide (NEM), which inhibits both the release of Ca2+ from internal stores and the influx of Ca2+, totally prevents the inhibition of PS synthesis induced by PHA, anti-CD3 or anti-CD2 mAbs. The presence, in the incubation medium, of either NDGA, TPCK or TPA, three drugs able to markedly inhibit Ca2+ influx without modifying the release of Ca2+ from internal stores, did not modify the inhibition of PS synthesis induced by PHA. Moreover all the drugs known to interact with calmodulin were also found to prevent the PHA-induced inhibition of this phospholipid. Taken together, these results show that the inhibition of PS synthesis induced by T cell activators is regulated by both calmodulin and Ca2+ ions recruited from intracellular compartments.