5,8,11,14-eicosatetraynoic acid (ETYA), an isomorphic competitive analogue of arachidonic acid, spontaneously generates a chemiluminescence signal detected with a liquid scintillation spectrometer operated at ambient temperature in the out-of-coincidence mode. The intensity of the signal was 10- or more-fold above background, required oxygen for its generation, was inhibited by antioxidants, and approximately doubled in D2O. Arachidonic acid, which contains 4-alkene rather than alkyne bonds did no more than double the chemiluminescent signal above background. When examined at 37 degrees C in a Berthold AutoLumat 958 luminometer, DBA (lucigenin) was required to detect a signal above background. Catalase or peroxidase, and to a lesser extent mannitol or histidine but not superoxide dismutase, strongly diminished the signal intensity. These observations provide a baseline for interpreting the functional and electron microscopic changes produced by ETYA in PC3 prostate and A172 glioblastoma cell lines, consistent with a contribution from oxidative stress associated with free radicals, and the absence of these morphological changes in U937 monoblastoid cells.