The innervation of the cat lower oesophagus, including the lower oesophageal sphincter, was studied by enzyme histochemistry, immunohistochemistry, and confocal microscopy. In the lower oesophageal sphincter, and at a level 2 cm above it, no apparent differences were seen in the nerve distribution pattern. Among the nerve populations studied, acetylcholinesterase (AChE)-positive nerves were the most abundant in both these regions. The density of AChE-positive nerves was particularly marked in the circular muscle layer. A rich supply of nitric oxide synthase (NOS)-containing nerves was identified by using an antiserum against neuronal NOS, or by enzyme histochemical staining for NADPH diaphorase activity. Vasoactive intestinal peptide (VIP)-immunoreactive nerves had a similar distribution pattern as NOS-immunoreactive nerves, and nerves displaying immunoreactivity for NOS and VIP often showed profiles coinciding with AChE-positive nerves. As judged by confocal microscopy, immunoreactivities for helospectin, pituitary adenylate cyclase-activating peptide (PACAP) and VIP, to a large extent were found in the same nerves. At a level 7 cm above the lower oesophageal sphincter, the total nerve supply was less than in the sphincter itself and 2 cm above it. Immunoreactivity towards VIP, PACAP and helospectin was also found to co-exist with NOS and neuropeptide Y within the same nerve structures. It is concluded that there is an intricate innervation pattern in the feline lower oesophagus reflecting the complexity in the regulation of its motility.