The effects of a mixture of peptidase inhibitors, consisting of 100 nM each of amastatin, phosphoramidon, and captopril, on the twitch inhibitory response (0.1 Hz, 0.5 ms duration, maximum intensity) exerted by opioid peptides were investigated. The opioid peptides, Met-enkephalin (50-200 nM), dynorphin(1-13) (0.2-1 nM), and beta h-endorphin (20-100 nM) concentration-dependently inhibited the electrically evoked twitch response. In the presence of the mixture of peptidase inhibitors, the twitch inhibition evoked by Met-enkephalin was significantly increased; however, the twitch inhibition evoked by beta h-endorphin and dynorphin(1-13) was only slightly increased. These increases were abolished by naloxone (NLX; 100 nM). Inhibition of the twitch response (0.1 Hz, 0.5 ms duration, maximum intensity) induced after high frequency stimulation (10 Hz, 0.5 ms pulse width, maximum voltage for various lengths of time) (post-tetanic twitch inhibition) was investigated in isolated guinea pig ileal longitudinal muscles. The mixture of peptidase inhibitors, which did not affect the twitch response or ACh-contraction, increased post-tetanic twitch inhibition. This increase was abolished by naloxone (100 nM). These results suggested that the potentiated post-tetanic twitch inhibition evoked by the peptidase inhibitors in the ileum was due primarily to mu-ligand(s) rather than to the kappa-type of endogenous opioid ligand(s) released from opioidergic neurons.