We have determined the effects of three radiographic contrast media on platelet aggregation and degranulation in vitro. Aggregation was measured as loss of single platelets, and degranulation was measured as P-selectin expression using flow cytometry. Iopamidol added to hirudinized blood induced aggregation directly and also potentiated that induced by weak platelet agonists such as adenosine diphosphate (ADP). Iodixanol also potentiated platelet aggregation, but ioxaglate inhibited it. Iopamidol also caused marked platelet degranulation. The pro-aggregatory effect of iopamidol was evident in non-anticoagulated blood as well as in hirudinized blood, but not in citrated blood. In platelet-rich plasma (PRP) prepared from hirudinized blood neither iopamidol nor iodixanol directly induced platelet aggregation, but they rendered platelets hypersensitive to ADP. ADP antagonists inhibited the platelet aggregation and degranulation induced by iopamidol in whole blood, whereas aspirin, an inhibitor of thromborane A2 synthesis, did not. These data are consistent with clinical reports of increased thromboembolic risk with non-ionic low-osmolar media, and raise concerns about the routine use of these contrast media during diagnostic and interventional arteriographic procedures. Routine use of citrate in previous experiments may have masked a pro-aggregatory effect of some contrast media.