Processing of the proenkephalin molecule will result in peptide fragments in which the pentapeptide YGGFM is included. We have employed a molecular sieve (2 kDa) separation, enzyme hydrolysis radioimmunoassay (RIA) treatment sequence which permits concurrent measurement of Met-enkephalin (Enk) and several enkephalin-encrypting (X-Enk) peptides in a single sample. Using this protocol, the release of Enk and X-Enk (total Enk-Enk) greater and less than 2 kDa from spinal cord and the mesencephalic aqueductal grey was assessed under resting conditions and during stimulation of the sciatic nerve in the chloralose-urethane anesthetized cat. Under resting conditions measurable levels of Enk (10.5 +/- 4.7; 9.1 +/- 2.1 pg/min) and X-Enk (47.8 +/- 19.7; 45.7 +/- 12.3 pg/min) are found in aqueductal and spinal superfusates, respectively. The X-Enk measured under resting and evoked conditions in aqueductal and spinal perfusates is associated almost exclusively (greater than 90-95%) with fragments greater than 2 kDa in size. There results, showing the relative absence of detectable levels of X-Enk forms greater than 2 kDa, were confirmed by reverse phase chromatography. During sciatic nerve stimulation, the levels of both Enk and X-Enk were mildly elevated in spinal and ventricular perfusates. With the addition of thiorphan (10(-5) M), though there was no effect on the resting release of either Enk or X-Enk, the levels of Enk measured under evoked conditions were significantly augmented in both ventricular and spinal perfusates.