When freshly drawn, heparinized human whole blood is incubated with 50 microM calcium ionophore A23187, platelets are stimulated to produce thromboxane B2 (TxB2) by activation of prostaglandin G/H synthase-l (PGHS-1). TxB2 concentration, as measured by immunoassay, is maximal at 20-30 min and declines thereafter. Addition of acetylsalicylic acid (IC50 = 2.8 microM) or other nonsteroidal antiinflammatory drugs (NSAIDs) 15 min or 4.5h prior to 30 min stimulation with ionophore results in concentration dependent inhibition of TxB2 production. When blood is incubated with 0.01-10 micrograms/ml E. colilipopolysaccharide (LPS), PGHS-2 is induced and TxB2 levels become detectable at 3h and continue to increase through 24h. Using a 5h incubation with 10 micrograms/ml LPS, aspirin (10 microM added at 0 h), which is rapidly metabolized to salicylic acid, had no effect on 10 micrograms/ml LPS-induced TxB2, but inhibited TxB2 production by ionophore A23187 added at 4.5h through acetylation of pre-existing PGHS-1. In a 5h assay, NSAIDs added at 0 h were compared for inhibition of TxB2 production stimulated by addition of ionophore A23187 at 4.5h (PGHS-1), or by addition of LPS at 0 h (PGHS-2). Most NSAIDs were more potent against PGHS-1 than PGHS-2. Diclofenac, naproxen and flufenamic acid were equipotent or slightly selective for PGHS-2. Diflunisal and nimesulide were > 4-fold selective for PGHS-2, and NS-398 was > 30-fold selective for PGHS-2.