The selectivity and potency of two new enkephalin-derived δ-opioid receptor agonists, DSTBULET ([D-Ser2(O-t-butyl), Leu5]enkephalyl-Thr6) and BUBU ([D-Ser2(O-t-butyl),Leu5]enkephalyl-Thr6 (O-t-butyl)) were determined with functional tests in vitro of μ-, δ-, and κ-opioid receptor activation in the rat brain. Both peptides concentration dependently (1 nM-1 μM) inhibited the release of radiolabeled acetylcholine (ACh) from striatal slices (pD2 7.6-7.9), an effect exclusively mediated by δ-opioid receptor activation. Fentanyl isothiocyanate (FIT), an irreversible δ-antagonist, completely blocked the inhibitory effects of DSTBULET and BUBU. Up to a concentration of 1 μM, the peptides did not affect striatal [3H]dopamine (DA) release nor cortical [3H]noradrenaline (NA) release, processes which are known to be inhibited by opioids activating κ and μ-receptors, respectively. Furthermore, both DSTBULET and BUBU caused a strong inhibition (pD2 8.2-8.3) of D-1 dopamine receptor-stimulated cyclic AMP efflux from striatal slices, an effect known to be mediated by μ- and/or δ-opioid receptor activation. However, the peptides were without effect when D-1 and D-2 dopamine receptors were stimulated simultaneously, a situation in which only μ-agonists are able to inhibit the resulting cAMP efflux. In conclusion, DSTBULET and BUBU appear to display a high selectivity and potency toward functional δ-opioid receptors in the brain.