Several pharmacological and electrophysiological studies have shown that the opioid receptors are widely distributed in the gastrointestinal tract. Despite such consensus, there are conflicting findings regarding their effects in intestinal function, and their precise site of action remained unclear. The aim of the present study was therefore to delineate the cellular localization of μ and κ opioid receptors in rat gastrointestinal tract using polyclonal antibodies generated to C-terminal end of the cloned μ (63 amino acids) and κ (41 amino acids) receptors. The distribution of μ differs from that of κ receptors within the gastrointestinal wall, with a greater abundance of μ receptor-like immunoreactive fibres in all intestinal layers. Numerous neurons expressing μ receptor-like proteins were found in the submucosal plexus with comparatively few in the myenteric plexus. In contrast, a higher number of neurons expressing κ receptor-like immunoreactivity were visualized in the myenteric plexus with a small number in the submucosal plexus. A high number of immunopositive neurons were found in the myenteric plexus of the stomach and the proximal colon with both antibodies. In the submucosal and mucosal layers, μ receptor-immunoreactive fibres were more abundant and distributed around the crypts, blood vessels and lymphatic nodes. Interestingly, numerous μ and fewer κ receptor-immunoreactive interstitial cells are localized in the region of myenteric plexus and at the internal border of the circular muscle. Finally, smooth muscle cells did not demonstrate any μ- nor κ-receptor immunoreactivity. These findings suggest that in the rat gastrointestinal tract, μ and κ opioid receptors may directly influence neuronal and interstitial cell activity. This appears not to be the case for the smooth muscle cells. In the muscular layers, the anatomical data point to μ receptor actions being mediated by nerve terminals, whereas κ receptor effects may be mediated by both nerve terminals and somatodendritic synaptic mechanisms. In contrast, in the submucosal and mucosal layers, μ receptors predominate and are localized on both nerve terminals and somatodendritic synaptic elements.