The neuropeptide eclosion hormone acts directly on the nervous system of the tobacco hornworm, Manduca sexta, to trigger ecdysis behavior at the end of each molt. Previous studies have shown that the action of eclosion hormone is mediated via the intracellular messenger cyclic GMP. In the present study we have investigated the mechanisms involved in the eclosion hormone-stimulated increases in cyclic GMP. No stimulation of guanylate cyclase was seen in homogenized nervous tissue, suggesting that eclosion hormone does not directly stimulate a membrane-bound form of guanylate cyclase. Nitric oxide synthase inhibitors, N-methylarginine and nitroarginine, had no effect on eclosion hormone-stimulated cyclic GMP levels. By contrast, 4-bromophenacyl bromide, an inhibitor of arachidonic acid release, and nordihydroguaiaretic acid, an inhibitor of arachidonic acid metabolism, almost completely abolished the eclosion hormone-stimulated cyclic GMP increase. We hypothesize that eclosion hormone receptors are coupled to a lipase, activation of which causes the release of arachidonic acid. Either the arachidonic acid directly stimulates the soluble guanylate cyclase or further metabolism of arachidonic acid yields compounds that activate guanylate cyclase.