Degradation of enkephalin by aminopeptidases has been established as an important functional mechanism that terminates the pharmacological action of enkephalins in the guinea-pig ileum. Aminopeptidases are a family of enzymes and little is known regarding the specificity of individual enzymes with respect to the degradation of enkephalins. Puromycin is a general inhibitor of aminopeptidases and bestatin is a more selective inhibitor of Leu-aminopeptidases and aminopeptidase B. Both agents are capable of inhibiting enkephalin degradation in broken cell preparations from brain. However, only bestatin enhanced the pharmacological response to enkephalin in the guinea-pig ileum and ileal longitudinal muscle. Bestatin enhanced the response to enkephalin in a concentration-dependent fashion. Furthermore, bestatin also decreased the formation of [3H]Tyr and increased [3H]Leu-enkephalin content after incubation of the guinea-pig ileum with [3H]Leu-enkephalin. In contrast, puromycin did not shift the concentration-response curve to Met-enkephalin in either the intact guinea-pig ileum or the ileal longitudinal muscle and, likewise, no alteration in the degradation of [3H]Leu- or Met-enkephalin occurred with puromycin. A small enhancing effect of puromycin on the duration of the inhibitory effect of enkephalin was observed only in the guinea-pig longitudinal muscle. This enhancement cannot be explained by an effect on enkephalin degradation, but may be related to some other action of puromycin. These data support the importance of aminopeptidase activity to the degradation of enkephalin and indicate that enzymes which have properties in common with Leu-aminopeptidases rather than arylamidases may be the primary aminopeptidases responsible for terminating the pharmacological actions of enkephalins in intact guinea-pig ileal preparations.