A series of diastereomeric dipeptides, analogues of the analgesic compound H-Lys-Trp(Nps)-OMe (2), containing 3,7-diamino-2-hydroxyheptanoic acid (DAHHA) and 2-[(o-nitrophenyl)sulfenyl]tryptophan [Trp(Nps)] has been synthesized. These compounds were tested as enkephalin-degrading aminopeptidases (APs), AP-M and AP-B inhibitors, and analgesics. The inhibitory potencies and the antinociceptive effects depended on the stereochemistry of the compounds. (2S,3R)-DAHHA-L-Trp(Nps)-OMe (26d) was a highly potent and selective enkephalin-degrading APs inhibitor, with an IC50 value in the 10(-8) M range. Although this derivative was about 10(3)-fold more potent than 2 against these enzymes, their antinociceptive effects were completely similar. These results indicate that the inhibitory capacity of this series of Trp(Nps)-containing dipeptides against enkephalin-degrading enzymes is not an important factor for their antinociceptive effects.