We examined the effect of acetaminophen and the structural analogues 2,6-dimethylacetaminophen, 3,5-dimethylacetaminophen, and N-acetyl-p-benzoquinone imine on human platelet aggregation, 14C-serotonin secretion, and thromboxane B2 synthesis. Preincubation with 1 mM acetaminophen for 2 min completely inhibited arachidonic acid- and collagen-stimulated platelet aggregation. Thromboxane B2 production and 14C-serotonin secretion by arachidonic acid-stimulated platelets also were completely inhibited. Preincubation of platelets with 1 mM 3,5-dimethylacetaminophen inhibited collagen and arachidonic acid-induced aggregation and arachidonic acid-stimulated thromboxane B2 synthesis, while treatment with 2,6-dimethylacetaminophen did not inhibit aggregation and blocked thromboxane B2 formation to a much lesser degree. Preincubation with 1 mM N-acetyl-p-benzoquinone imine inhibited arachidonic acid-induced aggregation and 14C-serotonin secretion but had no effect on arachidonic acid-induced thromboxane B2 formation and collagen-induced platelet aggregation.