Abstract The activity in cerebrospinal fluid (CSF) of dynorphin-converting enzyme (DCE) has been studied after voluntary exercise in the spontaneously hypertensive (SH) rat. The exercise consisted of spontaneous running in wheels for 4–5 weeks and the average running distance during the last two weeks was 4.0 km/24h. CSF samples were obtained under anaesthesia from the cisterna magna after penetration of the atlanto-occipital membrane. DCE transforms the members of the dynorphin family of opioid peptides into Leu-enkephalin-Arg 6. In the present investigation a radioimmunoassay was used for quantitation of Leu-enkephalin-Arg 6 release from dynorphin A 1–17 and dynorphin B 1–13. The rats that were running had a DCE activity (vs. both substrates) in CSF that was approximately 6–12 times higher than in animals not given the running opportunity. A statistically significant correlation between the two prodynorphin-derived substrate peptides was found in terms of DCE activity. We therefore propose that a single enzyme activity may be responsible for the hydrolysis of dynorphin B 1–13 and dynorphin A 1–17. Furthermore, a significant correlation was also found between running activity and DCE activity 12–14 h before the CSF was withdrawn. Besides measurement of DCE activity by radioimmunoassay, the formation of Leu-enkephalin-Arg 6 was identified by reversed-phase micro-column liquid chromatography and plasma desorption mass spectrometry. The experiment shows that voluntary exercise affects opioid peptidergic mechanisms.