Bovine aortic endothelial cells in culture were incubated with endotoxin. The amount of thromboxane A2 synthesized was then determined by a specific radioimmunoassay for thromboxane B2. After a lag of several hours the cells changed their shape and parallel to the change in cell shape release of thromboxane B2 occurred. At 24 h the amount of thromboxane B2 generated in response to endotoxin was 200-fold above baseline. Thromboxane B2 generation could be blocked by aspirin and the specific thromboxane synthetase inhibitor UK 37248. The endotoxin effect was dependent on protein and RNA synthesis as evidenced by the inhibitory action of cycloheximide (1.5 microM) and actinomycin D (2 micron).