Thromboxane A2 (rabbit aorta-contracting substance) is a proaggregatory vasoconstrictive, oxygenated metabolite of arachidonic acid which was originally discovered in guinea pig lung perfusates during antigen-induced anaphylaxis. The specific stimuli which activate synthesis and the cellular source in the lung remain undefined. In order to study pulmonary thromboxane A2 (TXA2) synthesis, a cultured lung cell model has been used. Monolayer cultures of human diploid embryonic lung fibroblast (WI-38) metabolized exogenously supplied [14C]arachidonic acid to TXA2 as well as prostaglandin E2. Both were unequivocally identified by gas chromatography/mass spectrometry. Cellular phospholipids were labeled by preincubating cultures overnight with [14C]arachidonic acid. Release of thromboxane A2 into the culture fluid from these prelabeled cultures was stimulated by two phospholipase activating agents, mellitin and the calcium ionophore A23187. The lung cells also released TXA2 and prostaglandin in a dose-dependent fashion when treated with thrombin but not when exposed to trypsin. Bradykinin, an anaphylactic mediator in vivo, was a potent TXA2 releasing agent in this in vitro system whereas histamine was inactive. In addition, anaphylactic shock perfusates from guinea pig lung were shown to contain a factor (other than bradykinin) which activates fibroblasts TXA2 synthesis in these cultured lung cells. These experiments indicate that the lung fibroblast is probably a source of pulmonary thromboxane in vivo and that the cultured lung cell system described here is a useful model for defining the complex interactions of mediators of anaphylaxis and asthma.