Experiments with washed rabbit platelets demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml), that causes maximal aggregation and partial release of amine granule contents, also causes increased accumulation of [3H]inositol-labelled inositol trisphosphate (InsP3) in the presence of 20 mM-Li+. This concentration of Li+ was found to inhibit the degradation of inositol phosphates by phosphomonoesterases. This result indicates that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is degraded early after platelet stimulation with thrombin, although in a previous study we had found no decrease in amount. In the absence of Li+, the labelling of inositol bisphosphate (InsP2) increased more rapidly than that of InsP3, consistent with rapid degradation of InsP3 by phosphomonoesterase. After 30s the increase in InsP2 was augmented by Li+. This increase in InsP2 could have been due to increased degradation of phosphatidylinositol 4-phosphate or inhibition of breakdown of InsP2 to InsP with a lesser inhibition of breakdown of InsP3 to InsP2. The effect on InsP3 and InsP2 of stimulation of the platelets with 1.0 unit of thrombin/ml was comparable with the effect of the lower concentration of thrombin. Inositol phosphate (InsP) labelling did not increase in response to 0.1 unit of thrombin/ml, but increased when the platelets were stimulated with 1.0 unit of thrombin/ml. Whether the increase in InsP was due to increased degradation of phosphatidylinositol or a greater rate of breakdown of InsP2 to InsP than InsP to inositol cannot be determined in these experiments. These results indicate that degradation of PtdIns(4,5)P2 is an early event in platelet activation by thrombin and that formation of inositol phosphates and 1,2-diacylglycerol rather than a decrease in PtdIns(4,5)P2 may be the important change.