The aim of the present immunohistochemical study was to investigate the localization of neurokinin 1 receptor (NK1R) in rat esophagus and examine the relationship between NK1Rs and intrinsic cholinergic, nitrergic, or substance P (SP) neurons. NK1R immunoreactivity (IR) was observed on the nerve cell bodies in the myenteric ganglia throughout the esophagus, but not on striated muscles and smooth muscle cells of the muscularis mucosae. The frequency of occurrence of NK1R neurons was highest in the cervical esophagus and lowest in the lower thoracic esophagus. Considerable immunoreactivity was seen on the nerve cell surfaces and was also present in the cytoplasm of cell somas and in the initial part of the axons, but not in any other nerve fibers or terminals. Dogiel type I-like morphology was observed in some of the NK1R neurons; however, the majority exhibited polymorphic morphology. Double immunolabeling indicated that a majority (77%) of the NK1R neurons were immunoreactive for choline acetyltransferase (ChAT), while a minority (23%) were immunoreactive for nitric oxide synthase (NOS)-IR. Most of the NK1R neurons (92%) were innervated by the SP nerve fibers. Triple immunolabeling indicated that 70% of the NK1R neurons were associated with intrinsic SP nerve fibers (without CGRP-IR), 59% were associated with extrinsic SP nerve fibers (with CGRP-IR), and 35% were associated with both intrinsic and extrinsic SP nerve fibers. These results suggest that SP/tachykinin released from the SP nerve fibers of intrinsic and/or extrinsic origin activates the predominantly intrinsic cholinergic neurons via NK1Rs to influence neuronal transmission or motility in rat esophagus.