The effect of acute and chronic morphine treatment was investigated on the spontaneous and evoked release of methionine enkephalin-like immunoreactivity (MELI) in vivo using the technique of cat spinal superfusion and ventriculocisternal perfusion. Stimulation of sciatic nerve, at intensities known to activate small-diameter nerve fibers, resulted in a consistent release of MELI from the spinal cord and the brain. Local application of morphine (5 X 10(-4)M) to the spinal cord resulted in a significant decrease in the evoked release of MELI. Naloxone (2 mg/kg i.v.), administered during morphine treatment, produced an increase in the spontaneous and greatly augmented the evoked release of spinal and ventricular MELI. In morphine-naive animals, naloxone did not affect the spontaneous or evoked MELI release. In cats chronically exposed to parenteral morphine by implantation of morphine pellets (2 X 75 mg), the spontaneous release of spinal and ventricular MELI was significantly greater than this release in control animals. Stimulation of sciatic nerves evoked a normal release of MELI in morphine-pelleted animals. Administration of naloxone to these animals resulted in a large and sustained increase in the spontaneous release of MELI from the spinal cord and brain. The material released by stimulation was identified as methionine enkephalin-like on the basis of similar results with two different antisera, parallel displacement curves with serial dilutions of spinal and ventricular perfusates and comigration with methionine enkephalin on a Sephadex G-25 column. These results suggest that if there is a tonic suppression of enkephalin release mediated by opiate receptors, these receptors display a tolerance development also. The facilitated release by naloxone in the chronic morphine-treated animals may indicate that reversal of the ongoing opioid inhibition results either in an excessive drive of the enkephalinergic neuron by other excitatory systems or the loss of a tonic auto-inhibition, which is not present in the non-tolerant animal.