Intact segments of rat ileum were stimulated in vitro by either electrical field stimulation (EFS) or cholinergic stimulation with carbachol, in the presence of absence of varying insulin concentrations (50, 100 and 200 μU/ml), and the contraction in the longitudinal axis was recorded. Insulin had no significant influence on the carbachol contractile dose-response curve nor did it affect the cholinergically mediated ‘on-contraction’ at onset of the electrical stimulus. The ‘off-contraction’ occurring at cessation of the electrical stimulus was partly mediated by a cholinergic and partly by a noncholinergic and nonadrenergic mechanism, and decreased over time with repeated stimulation. This decay with time was significantly attenuated by insulin. In the presence of insulin, release of bombesin-like immunoreactivity induced by carbachol or EFS significantly increased. On the other hand, the release of somatostatin-like immunoreactivity was suppressed by 50–70% in the presence of insulin. These results demonstrate that in vitro insulin can modify both the motility responses of isolated segments of rat ileum and the neuropeptide release from suc