Human platelets and platelet particulate fractions were found to emit a burst of chemiluminescence during incubation with arachidonic acid. The magnitude of light emission was directly related to the number of platelets in the reaction mixture and varied little for the same individual from day to day. The chemiluminescence response of platelets was localized to the particulate fraction and was almost totally oxygen dependent. In addition to arachidonate, seven other polyunsaturated fatty acids, including several that are not prostaglandin precursors, also induced platelet chemiluminescence.A correlation was sought between chemiluminescence and platelet prostaglandin synthesis. Platelets incubated in low concentrations of aspirin, or platelets from subjects who had ingested aspirin, had markedly decreased arachidonic acid-induced chemiluminescence. Salicylic and sulfosalicylic acid had no inhibitory effect. A time-response curve of aspirin inhibition of arachidonate-induced chemiluminescence closely paralleled a time-response curve of aspirin inhibition of malondialdehyde production. Linoleic acid-induced platelet chemiluminescence was also markedly inhibited using aspirin-incubated platelets or platelets from subjects who had ingested aspirin. These studies implicate activation of the enzyme prostaglandin synthetase in the arachidonate-induced platelet chemiluminescence. They provide evidence that linoleic acid may also specifically activate platelet cyclooxygenase to produce electronically excited species capable of light emission.