Motilin, a 22-amino acid gastrointestinal peptide, and ghrelin, the natural ligand of the growth hormone secretagogue receptor, form a new group of structurally related peptides. Several lines of evidence suggest that motilin and ghrelin are involved in the control of gastrointestinal motility by the activation of receptors on enteric neurons. The aim of this study was to look for the existence of motilin, ghrelin, and their respective receptors in the myenteric plexus of the guinea pig. We used longitudinal muscle/myenteric plexus (LMMP) preparations and cultures of myenteric neurons of the guinea pig ileum, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR). Most of the motilin-immunoreactive (IR; 72.8%) and motilin receptor-IR (68.9%) neurons were also positive for neuronal nitric oxide synthase (nNOS), 72.8% and 68.9%, few for choline acetyl transferase (ChAT), 11.4% and 11.9%, respectively. In contrast, ghrelin was mainly colocalized with ChAT (72.2%), and only 3.6% of ghrelin-positive cells showed nNOS-IR in the LMMP. Neither motilin nor the motilin receptor or ghrelin colocalized with calbindin. RT-PCR studies revealed motilin, ghrelin, and ghrelin receptor mRNA transcripts in LMMP preparations and in cultured myenteric neurons. In conclusion, this study, for the first time, provides direct evidence for the existence of motilin and ghrelin in myenteric neurons and suggests that both peptides may play a role in the activation of the enteric nervous system and hence in the regulation of gastrointestinal motility.