Recent studies have demonstrated that unsaturated fatty acids are involved in the regulation of neuroeffector function. I have extended these studies by examining the effect of arachidonic acid on neuromuscular function in vitro using the rat phrenic nerve-diaphragm preparation. Arachidonic acid caused a time-and dose-dependent reduction in indirectly stimulated twitch tension, but had no effect on directly stimulated twitch tension. Linoleic acid and linolenic acid also reduced indirectly stimulated twitch tension, whereas stearic acid, oleic acid and arachidic acid had no effect. None of three blockers of arachidonic acid metabolism, the cyclooxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguaiaretic acid or the cytochrome P-450 inhibitor ketoconazole, altered the effect of arachidonic acid on twitch tension. The free radical scavenger superoxide dismutase eliminated the inhibitory effect of arachidonic acid on twitch tension, suggesting that superoxide anion played a role in arachidonic acid's action.