Incubation of [3H]-tyrosine methionine5-enkephalin (MET) with human brain preparations (100,000g supernatant; sections of the limbic system, thalamus, basal ganglia, cerebellum, and cortex) results in its rapid and complete degradation; over 95% of the initial labeled tyrosine is recovered as the free aminoacid within 10 min. Results show a considerable range in the peptide initial velocity (Iv) and half-life (t1/2) degradation values obtained from different brain sections of individual brains, either from the same or from different main brain areas. This relatively wide range of values was scattered, failing to identify consistent differences between the various brains areas studied. Differences in brain tissue storage time or repeated sample freezing and thawing failed to alter significantly either of these kinetic parameters of MET metabolism. Peptide degradation rate (optimum pH and temperature of 7.4 and 37°C, respectively) was concentration-dependent inhibited by known aminopeptidase inhibitors (puromycin, bacitracin, and bestatin, and to a lesser extent by thioridazine). However, it was not significantly affected by either N-carboxymethyl phenyl leucine, captopril or thiorphan [dipeptidyl peptidase(s) or peptidyl dipeptidase(s) inhibitors, respectively]. A better understanding of the mechanisms regulating brain MET metabolism may contribute to the rational design of pharmacological strategies based in the modulation of its bioavailability. © 2007 Springer Science+Business Media, LLC.