Platelet aggregation and secretion induced by phorbol ester (PMA) or diacylglycerol (DAG) are preceded by an increase in [Ca++] that is detected byaequorin, but not by quin2, fura-2, or indo-1, suggesting that these indicatorsreflect different aspects of Ca++ homeostasis, possibly different functional Ca++ pools. Addition of two conventional agonists in subthreold concentrations synergistically enhances the [Ca++] rise and aggregation.However, if PMA or DAG is the first agonist the subsequent quin2-indicated [Ca++] rise after thrombin is reduced.Whether aequorin-indicated [Ca++] is similarly affected is unknown. We studied gel-filtered platelets loaded with aequorin or a fluorophore and added PMA, DAG, thrombin or ADP, alone or in combination. Either PMA or DAG alone caused a concentration-dependent increase in [Ca++] detectable with aequorin but not with the fluorophores; simultaneous addition of thrombin or ADP with DAG or PMA produced a larger [Ca++] rise than either alone. However, addition of DAG or PMA as a first agonist reduced subsequent aequorin-indicated [Ca++] rises following thrombin or ADP in a concentration and time-dependent manner. Inhibition of ADP or thrombin-induced [Ca++] rise was not always accompanied by inhibition of aggregation or secretion. Combination of subthreshold concentrations of ADP and thrombin produced an enhanced [Ca++] rise and aggregation. However, this synergistic effect was inhibited by preincubation with DAG or PMA. Neither this effect nor DAG-induced [Ca++] rise was inhibited by the protein kinase C inhibitor H-7. In genera^ preincubation of platelets with an agonist enhances Ca rise and aggregation in response to a second agonist; in contrasl protein kinase C activators, which themselves elevate [Ca++] as shown by aequorin, inhibit aequorin-indicated Ca rises after ADP or thrombin, and limit synergism between these two agonists.