The inhibition of the enzymatic degradation of methionine enkephalin (Met-Enk) was investigated kinetically in nasal, rectal, and vaginal extracts of rabbits with and without inhibitors, such as puromycin (PM), amastatin (AM), thiorphan (TP), Na 2EDTA, and thimerosal (TM), alone or in combination, by analyzing the parent peptide and its hydrolytic fragments by HPLC. The effects of variation of pH in the nasal extracts, and the addition of 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CyD) on the stabilization of Met-Enk were also studied. The degradation of Met-Enk was found to be fastest at around pH 7, indicating that the activity of enkephalin-degrading enzymes is optimal at this pH. Addition of 2-HP-β-CyD (10%) to the nasal, rectal, and vaginal extracts was noted to reduce the first-order degradation rate constants for Met-Enk by 2.5-2.8-fold, compared to the control. AM alone inhibited the enzymatic degradation of Met-Enk with IC 50 values of 3.5 and 0.22 μM for the rectal and vaginal extracts, respectively, whereas PM was found to be approx. 14.2- and 26.8-fold less potent than AM, respectively. The effects of both aminopeptidase inhibitors in the nasal extracts were smaller. Even at 50 μM, TP (a potent enkephalinase A inhibitor) alone revealed only a small increase of Met-Enk stability in the various mucosal extracts, however, EDTA (5 μM) was observed to inhibit enzymatic hydrolysis considerably by blocking both enkephalinase A and B and, to some extent, aminopeptidase. On the other hand, TM (0.05%) was found to be a new and potent inhibitor for enkephalinase B and aminopeptidases, which was more potent than AM (50 μM) in inhibiting the degradation of Met-Enk in various mucosal extracts. Furthermore, the addition of TM (0.01%) to a combination of AM (50 μM) and EDTA (5 μM) was observed to protect Met-Enk from enzymatic degradation in nasal, rectal, and vaginal extracts by more than 90%, after 24 h of incubation, by inhibiting almost completely all the enkephalin-degrading enzymes present in the incubation mixtures.