To better understand the local neuronal network of the gastro-duodenal junction in ruminants, we identified the components of the enteric nervous system (ENS) innervating the pyloric sphincter (PS) of the lamb abomasum. The neurons were labelled after injecting the tracer Fast Blue (FB) into the wall of the PS, and the phenotype of the FB-labelled neurons was immunohistochemically investigated using antibodies against nitric oxide synthase (NOS), choline acetyltransferase (ChAT), tachykinin (substance P) and tyrosine hydroxylase (TH). The FB-labelled abomasal myenteric plexus (MP) neurons, observed up to 14cm from the PS, were NOS-immunoreactive (IR) (82+/-12%), ChAT-IR (51+/-29%), SP-IR (61+/-33%), and also TH-IR (2%). The descending nitrergic neurons were also SP-IR (64%) and ChAT-IR (21%); the cholinergic descending neurons were SP-IR (3%). The FB-labelled duodenal neurons were located only in the MP, up to 8cm from the sphincter and were ChAT-IR (79+/-16%), SP-IR (32+/-18%), NOS-IR (from 0 to 2%), and also TH-IR (4+/-3%). The cholinergic ascending neurons were also SP-IR (60%) whereas no ChAT-IR cells were NOS-IR. The findings of this research indicate that the sheep PS is innervated by long-projecting neurons of the abomasal and duodenal ENS.