The rabbit iris-ciliary body contains 78 +/- 6 ng/gm serotonin (5-HT) while the amine content in the aqueous humor is less than 0.01 ng/100 microliter. The low levels of endogenous 5-HT in the iris-ciliary body could not be directly detected by immunocytochemistry. However, pretreatment in vivo and in vitro with L-tryptophan and pargyline resulted in the localization of a sparse population of 5-HT fibers. These fibers could not be studied by exposing the tissue to exogenous 5-HT since the amine was taken up by noradrenergic fibers as well. This was confirmed in studies involving superior cervical ganglionectomy. It is concluded that 5-HT is taken up by both serotonergic and adrenergic fibers of the iris-ciliary body. In the presence of lithium, 5-HT stimulated an increase in the 3H-inositol phosphate accumulation in a dose-dependent manner in tissue where the phosphoinositide pool was labeled with 3H-inositol. A variety of agonists and antagonists were used to show that the 5-HT response is mediated, at least partly, by 5-HT2 receptors. The 5-HT-mediated effect on inositol phosphates is unaffected by superior cervical ganglionectomy. The effect of noradrenaline, which also stimulates inositol phosphate accumulation (but via alpha 1-adrenergic receptors in the iris-ciliary body), was elevated following superior cervical ganglionectomy.