Murine T helper cloned cells permeabilized with the bacterial lysin, tetanolysin, were used to investigate the role of intracellular Ca2+ in regulating myo-[2-3H] inositol phospholipid (InsPL) hydrolysis triggered upon perturbation of the T cell receptor-CD3 complex. [Ca2+] was controlled by a calcium/magnesium/EGTA buffer. Antibody (mAb) aggregation of CD3 induced InsPL hydrolysis in the absence of added Ca2+. However, stimulated InsPL hydrolysis increased with the free [Ca2+], reaching a maximum at 100-300 nM [Ca2+]. Ca2+ increased the overall efficiency of hydrolysis without changes in EC50 of the anti-CD3 mAb. The response diminished at > 300 nM [Ca2+] due to a mixed type inhibition. Ca2+ alone had no effect on inositol phosphate levels. Polyphosphoinositides were preferentially cleaved, since no accumulation of Ins(1)P/Ins(3)P was detected, indicating that direct hydrolysis of phosphatidylinositol did not occur, irrespective of the Ca2+ concentration. [Ca2+] above 300 nM shifted the relative amounts of CD3-induced inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) in favor of the latter. Unlabeled permeabilized cells exposed to > or = 100 nM [Ca2+] showed enhanced conversion of [3H]Ins(1,4,5)P3 to [3H]Ins(1,3,4,5)P4. In conclusion, InsPL hydrolysis is optimally triggered by CD3 perturbation at intracellular Ca2+ levels approximating those observed in intact resting lymphocytes (100 nM). Ca2+ concentrations similar to those triggered by InsPL-derived metabolites may inhibit InsPL hydrolysis and promote Ins(1,3,4,5)P4 production, thus controlling the amounts of Ins(1,4,5)P3.