Using a combination of solid-phase and solution methods, we synthesized a series of cyclic [Leu5]enkephalin analogues by substitution of D-alpha, omega-diamino acids in position 2 of the enkephalin sequence and cyclization of the omega-amino group to the C-terminal carboxy group of leucine. Cyclic analogues containing D-alpha, beta-diaminopropionic acid (1), D-alpha, gamma-diaminobutyric acid (2), D-ornithine (3), or D-lysine (4) in position 2 and the [D-Leu5] and [des-Leu5] analogues of 4 (5 and 6) showed, in general, high potency in the guinea pig ileum (GPI) assay and low potency in the mouse vas deferens (MVD) assay. IC50 (MVD)/IC50 (GPI) ratios ranging from 3.1 to 29.4 were obtained, indicating the preference of the cyclic analogues for mu receptors over delta receptors. With two exceptions, preferential affinity for mu receptors is reflected in the Ki ratios determined in parallel binding assays using [3H]naloxone and [3H] [D-Ala2, D-Leu5]enkephalin as mu and delta receptor selective radioligands, respectively. Comparison of the pharmacological profiles of the cyclic analogues 1-4 with those of their corresponding open-chain analogues, [D-Ala2, Leu5]enkephalinamide (1a), [D-Abu2, Leu5]enkephalinamide (2a), [D-Nva2, Leu5]enkephalinamide (3a), and [D-Nle2, Leu5]enkephalinamide (4a), revealed that the pronounced mu character of compounds 1-4 is a direct consequence of the conformational constraints introduced by cyclization. This finding is in agreement with the concept of different conformational requirements of mu- and delta-opiate receptors and raises the possibility of manipulating opiate receptor selectivity by varying the type and degree of conformational restriction.