The relationships between various properties of inhibitors of enkephalinase (membrane metalloendopeptidase, EC 3.4.24.11) i.e., enzyme inhibition, protection of endogenous enkephalins, antinociceptive activity and stimulation of locomotor activity was investigated by comparing the relative potencies of the two enantiomers of Thiorphan and acetorphan, its parenterally active prodrug. In vitro (R)- and (S)-Thiorphan were almost equipotent in inhibiting enkephalinase activity (Ki, 1.7 and 2.2 nM, respectively) or thermolysin activity (Ki, 13 and 6 microM, respectively) whereas the (R)-isomer was 44-fold less potent than the (S)-isomer on ACE activity (Ki 4800 and 110 nM, respectively). When tested on slices of rat globus pallidus in the presence of bestatin, to block the aminopeptidase pathway of enkephalin degradation, both Thiorphan enantiomers ensured a complete protection of endogenous (Met5)enkephalin released by depolarization and a suppression of the increase in the extracellular levels of Tyr-Gly-Gly, a characteristic enkephalin metabolite. These two effects occurred at EC50 values of the two enantiomers (10 nM in both cases), consistent with the idea that they were due to enkephalinase inhibition. After i.v. administration of the acetorphan enantiomers to mice, the enkephalinase activity of a rapidly prepared striatal membrane fraction was reduced in a dose-dependent manner with similar "ex vivo" ED50 values (1.0 and 0.3 mg/kg for the (R)- and (S)-isomer, respectively). In contrast the ACE activity of the same preparation was reduced in a significant manner only by (S)-acetorphan (ED50 value of 11 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)