Abstract Antibody-coated microprobes were used to determine whether immunoreactive enkephalins were released in response to fatiguing isometric contractions of the hind-limb muscles in cats anesthetized with α-chloralose. Contractions were performed by stimulating the tibial nerve via a microprocessor-controlled stimulator. Microprobes were inserted into the periaqueductal grey (P 0.5–1.0 mm) prior to, during and following fatiguing contractions. During fatiguing contractions, mean arterial blood pressure increased by 76 ± 9 mmHg above resting and recovery levels. Levels of immunoreactive enkephalins were elevated in the dorsolateral periaqueductal grey during the isometric contraction when compared to resting levels. It is possible that isometric muscle contraction causes the release of Met-enkephalin-like substances in the periaqueductal grey.