Contrary to a recent report [Rinder et al.: Blood 82: 505, 19931, aspirin does inhibit the release of a-granule contents as well as inhibiting the release of dense granule contents by human platelets during ADP-induced aggregation in citrated platelet-rich plasma (PRP). Measurements were: percent release of 14C-serotonin from prelabeled platelets, radioimmunoassay of p-thromboglobulin (PTG), and expression on the platelet surface of the a-granule constituent, P-selectin, by flow cytometry. During the second phase of ADP-induced aggregation, 69.0 2 8.3% of pTG and 54.1 f 4.6% of 14C-serotonin were released (means 2 SEM, n= 13); aspirin treatment reduced these values to 6.0 f 1.2 and 1. O 0.3%, respectively. In contrast, incubation of platelets with ADP without stirring caused only 6.7 & 1.7% release of pTG and 2.1 2 0.4% release of’‘C-serotonin; these low values were not appreciably affected by aspirin. During ADP-induced primary aggregation in PRP anticoagulated with FPRCH, CI (PPACK), only 4.7 2 0.9% release of PTG and no detectable release of 14C-serotonin occurred; aspirin had no effect. In both stirred and unstirred PRP, the thrombin receptor activating peptide, SFLLRN (50 pM), caused at least 75% release of the contents of both granules, which was partially inhibited by aspirin. Upon incubation of platelets with ADP (2-10 pM), the mean fluorescence intensity due to P-selectin was 44% of that induced by SFLLRN. In this unstirred system used for flow cytometry, aspirin treatment caused no significant inhibition of P-selectin expression. Thus, under conditions in which ADP does not cause secondary aggregation (physiological Ca2+ concentration or unstirred citrated PRP) release of the contents of both types of granules is less than 7% and aspirin is not inhibitory; the P-selectin expression associated with this low percent release is also unaffected by aspirin. However, aspirin does strongly inhibit the extensive release of both a-granule and dense granule contents during ADP-induced secondary aggregation in citrated PRP.