Evidence in supporting the presence as well as the absence of the cholinergic innervation of mammalian pineal glands have presented. However, it is still controversial. In the present study, five lines of experiments were investigated to prove the criteria of innervation and functions of cholinergic system on the bovine pineal gland. The biochemical assay of choline acetyl-transferase (ChAT), enzyme for acetylcholine synthesis, was preformed including its kinetic study. The ChAT of bovine pineal gland had the specific activity of 0.0339(+or-)0.0042 nmo1/min/mg protein with the Michealis-Menten constant K(,m) values of 124.86(+or-)24.06 um and 3.11(+or-)0.94 mM with respect to acety1 CoA and choline, respectively and with thre maximum initial velocity V(,max) values of 0.0598(+or-)0.0034 nmol/min/mg protein and 0.0155(+or-) 0.0060 mno1/min/mg protein with respect to acetyl CoA and choline, respectively. By immunohistochemical localization technique using both monoclonal and polyclonal antibodies against choline acetyltransferase (ChAT) with avidin-biotin comples method, the CHAT-immunoreactive (IR) nerve figers were located throughout the pineal gland, both in the perivascular spaces and between the pinealocytes. In addition, some ChAT-IR intrapineal neurons were found. Within the rostral and caudal pineal stalk,ChAT-Ir nerve fibers were demonstrated and could be followed into the gland. At the caudal tip of the pineal gland, a bundle of ChAT-IR nerve fibers was observed to penetrate into the gland together with blood vessels. The muscarinic shalinergic receptors sites have been indentified and characterized in bovine pineal gland by using [(30H](-) quinuclidinyl benzolate (QNB) as a radioligand. AT 25Degree C, the binding reached equilibrium within 60 min and remained constant for an addition two hours. Furthermore, the binding was saturable, reversible and tissue dipendent in nature. The saturation experiment of muscarinic receptor sites revealed a single binding site with a dissociation equilibrium constant (K(,D)) of 0.423(+or-)0.01 nM and a receptor density (B(,max)) of 69.75(+or-)20.91 fmol/mg protein. Then, the concentrationof hte drugs required ton innibit 50% of the binding (IC(,50)) were determined having the descending order of potency in competing with ligand binding to be dexetimide>(+or-)QNB>scopolamine> atropine>pilocarpine>oxotremorine>levetimide>acetylcholine> carbachol. By in vitro autoradiographic receptor binding technique, using the [(3)H](-)QNB as a ligand, the localization of muscarinic receptor sites were demonstrated on the membrane of both pinealocytes and intrapineal neurons. Moreover, in order top indicate the function of the cholinergic system on the pineal gland, the effect of muscarinic agonists on the serotonin N-acetyltransferase (SNAT), a rate limiting enzyme of melatonin synthesis, activity was wxamined in the bovine pineal explant in culture. The muscarinic agonists, i.e. methachaline, pilocarpine, oxotremorine and carbochol significantly inhibit the SNAT activity. The inhibition was in dose-dependent fashion. In addition, the inhibitory effect of agonists was blocked by cholinergic antagonists, i.e. atropine sugate and (+or-)QNB. In conclusion, the pharmocological and anatomical evidences presented in this study strongly support the cholinergic innervation of the bovine pineal gland. In addition, by the inhibitory control of the cholinergic agonists on the SNAT activitly indicates a functional infuluence of a cholinergic mervous system on the pineal gland.