The superior cervical ganglion (SCG) was reinnervated by vagal afferent fibers by cross anastomosis between the cranial end of nodose ganglion and the caudal end of SCG in cats. Formation of functional synapses was evidenced by unilateral mydriasis and contraction of the nictitating membrane in response to inflation of the stomach with a balloon or to electrical stimulation of the afferent vagus. The acetylcholine (ACh) content in the cross-anastomosed SCG (reinnervated by vagal afferent fibers) was measured. In anastomosed SCG, the ACh content was about half of normal SCG, but significantly higher than chronically decentralized SCG. Also the ACh content in nodose ganglion (NDG) was investigated in situations in which there was anastomosis, chronic supra, infra, or supra-/infranodose vagotomy. The ACh content of anastomosed NDG was near that of supranosdose vagotomized ganglion. The ACh content of supra-/infranodose vagotomized NDG, which can be considered the NDG itself, was as much as that of normal intact NDG. It was found that the ACh content of infranodose vagotomized NDG was increased, possibly the result of vagal efferent axonal flow or transport. The ACh content of vagal trunk with or without infranodose vagotomy was also measured. The ACh content of vagal trunk with infranodose vagotomy was smaller than that of the normal trunk, but there was still a considerable quantity of ACh. There was no significant change in wet weight of the SCG and NDG before or after the operations. From these results we have concluded that the transmission of the cross-anastomosed SCG (reinnervated with vagal afferent nerve) was cholinergic; and that the vagal afferent nerve have afferent cell bodies not only in NDG but also in peripheral vagal trunks (infranodose portion). These results strongly suggest that vagal afferent fibers are in part cholinergic.