To elucidate the structural features determining δ-opioid receptor properties of enkephalin analogues containing Cys(O2NH2) in position 2, a series of Cys2-containing derivatives were synthesized and tested for their effectiveness in depressing electrically evoked contractions of the mouse vas deferens (predominantly enkephalin-selective δ-opioid receptors) and the guinea-pig ileum (μ- and κ-opioid receptors). The peptidase resistance of the compounds was also tested. The ratio IC50 in the guinea-pig ileum/IC50 in the mouse vas deferens, indicating selectivity for δ-opioid receptors, was high for Cys(O2NH2)2-containing analogues and especially for [Cys(O2NH2)2, Leu5]enkephalin, which was about seven times more selective than δ-opioid receptor selective ligand cyclic [d-Pen2, d-Pen5]enkephalin (DPDPE). The dissociation constant (KA) and relative efficacy (erel) of the compounds in the mouse-isolated vas deferens were determined using explicit formulae derived by fitting of the data points with two-parametric hyperbolic function. The obtained values for KA and erel suggest that: (i) incorporation of Cys(O2NH2)2 in the molecule of [Leu5]enkephalin highly increases the efficacy and does not change significantly the affinity of the respective analogues to δ-opioid receptors; [Cys(O2NH2)2, Leu5]enkephalin has higher affinity than DPDPE, but is less resistant to enzyme degradation; the effect of this modification on the efficacy is decreased when methionine is in position 5; (ii) d-configuration of Cys(O2NH2)2-containing analogues increases their peptidase resistance, but reduces efficacy and affinity of the peptides towards δ-opioid receptors; (iii) the substitution of Cys(O2NH2) with Hcy(O2NH2) reduces the efficacy, affinity and potency of the respective analogues and maintains their sensitivity to endogenous peptidases; (iv) the substitution of the sulfonamide group with benzyl group in the molecule of Cys in position 2 decreases their efficacy and affinity toward δ-opioid receptors, but attaches resistance to enzyme degradation. The results obtained in this study allow: (i) to involve the receptor affinity and agonist efficacy as drug-design consideration for δ-opioid receptor properties of newly synthesized compounds and (ii) to characterize some of the structural features, which set the pattern for their opioid profiles. [Copyright &y& Elsevier]