The thromboxane synthesizing system from human platelets has been characterized by using arachidonic acid and prostaglandin endoperoxide as indirect and direct substrates. The synthesis of thromboxanes from either substrates catalyzed by the microsomal fraction was monitored by measuring the formation of thromboxane B2 immunoreactivity. Both hemoglobin and phenolic compounds were required for the maximal synthesis of thromboxane B2 from arachidonic acid but not from prostaglandin endoperoxide. Studies on the kinetics of the formation of thromboxane B2 from either substrates indicated that the synthesis of prostaglandin endoperoxide intermediate is the rate-limiting step in the overall production of thromboxanes from arachidonic acid. Effect of the microsomal protein concentrations on the rate of formation of thromboxane B2 showed concave upward relationship suggesting the possible involvement of endogenous stimulator(s) in thromboxane synthetase catalyzed reaction. A variety of compounds including sulfhydryl inhibitors, prostaglandin endoperoxide analog, prostaglandin antagonist, N-0164, and N-substituted imidazoles could directly inhibit the thromboxane synthetase, while nonsteroidal antiinflammatory drugs apparently affected the prostaglandin endoperoxide synthetase which catalyzes the synthesis of the immediate precursor of thromboxanes.