The influence of halothane, or naloxone, or halothane followed by naloxone was investigated in the in vitro myenteric plexus longitudinal muscle preparation of the guinea pig ileum. Halothane alone in 1.5 to 2.0% (v/v) concentration caused about 50% depression of the twitch and decreased both spontaneous acetylcholine (ACh) release (p < 0.02) and volley output of ACh (p < 0.02). Very high concentrations (>1 μM) of naloxone caused a nonspecific, postsynaptic depression of the twitch. Higher than 100 nM concentrations of naloxone increased spontaneous ACh release, but had no effect on the volley output of ACh. Over a wide concentration range, from 15 nM to 3 μM, naloxone did not antagonize in the longitudinal muscle preparation the effects of halothane on any of the parameters investigated. These findings indicate that the sites of action of halothane and naloxone in this preparation are not identical.