We have compared the abilities of exogenously added U46619, the PG endoperoxide analogue and, sn-l-oleoyl 2-acetylglycerol (OAG) and sn-1,2-dioctanoylglycerol (diCg), the membrane-permeant DAG analogues, at restoring weak agonist-induced secretion in indomethacin (10μM)-treated platelets (I-PL) in the absence of endogenous PG/Tx synthesis. [14C]-5HT secretion from pre-loaded, washed human platelets was correlated with the levels of [Ca2+]i, using platelets loaded with quin 2. Concentrations of OAG (62-125μM) and diCg (15-30μM), which have previously been shown to be fully effective at activating protein kinase C, failed to significantly enhance [14C]-5HT secretion in combination with ADP (10μM), adrenaline (10μM) or PAF (0.2μM) although they potentiated platelet aggregation, when added 10-30 sec after these agonists to I-PL. eg ADP-0%, 30jiM diCg-9.8%, ADP+diCg-11.9%, 5HT release (p>O.05). In contrast, a low concentration of U46619 (0.2μM), that induced no aggregation, [14C]-5HT secretion or rise in [Ca2+]i levels on its own, was able to synergize strongly at potentiating secretion in combination with all three weak agonists examined, as well as in combination with OAG and diCg (U46619-0%, ADP+U46619-20.4%, U46619+30μM diC8-48% 5HT release) . The greater effectiveness of U46619 at potentiating secretion in combination with the weak agonists was not related to different degrees of [Ca2+]i mobilisation, as ADP and PAF-induced rise in [Ca2+]i occurred to a similar degree in the presence of U46619 and diCg. At a higher concentration of U46619 (0.6μM), which was maximally effective at inducing secretion and elevating [Ca2+]i levels on its own, addition of the weak agonists or OAG or diCg, along with U46619, resulted in a further enhancement of secretion which was independent of changes in [Ca2+]i levels. The results demonstrate that U46619 but not OAG or diCg, is able to fully restore weak agonist-induced secretion in indomethacin-treated platelets, suggesting that the actions of endogenously formed PG endoperoxides/TxA2 cannot be substituted by DAG and raised [Ca2+]i levels and, may be mediated via a mechanism additional to that involving these mediators.