This work explored the role of the cholinergic pathway, assessed at a post-synaptic level by the use of isolated smooth muscle cells, in the impairment of antral motility associated with diabetic gastroparesis. Contractile response to carbachol--but not to erythromycin, a motilin receptor agonist--was abolished in antral smooth muscle cells isolated from (i) rats previously rendered diabetic by a single i.v. dose of streptozotocin (STZ, 60 mg/kg) and (ii) db/db spontaneously diabetic mice. Insulin treatment of STZ-rats was able to prevent the impairment of the carbachol contractile response, but not to reverse it once established. In STZ-rats, impairment of contractile response was not associated with a change in density of [3H]-N-methyl-scopolamine ([3H]-NMS) binding sites (approximately 1.5 fmol/mg protein). Displacement curve of the [3H]-NMS binding by carbachol was shifted to the right in diabetic rats as compared to controls. The addition of GTP-gamma-S induced a shift to the right of the displacement curve in control but not in diabetic animals. These results strongly suggest that diabetes is associated with an early and specific alteration of the muscarinic control of contraction of antral smooth muscles at a post-synaptic level, associated with an alteration of the GTP-binding proteins coupled to muscarinic receptors.