Endomorphins were subjected to a number of structural modifications in a search for their bioactive conformations. The alicyclic beta-amino acids cis-(1 S,2 R)ACPC/ACHC, cis-(1 R,2 S)ACPC/ACHC, trans-(1 S,2 S)ACPC/ACHC, and trans-(1 R,2 R)ACPC/ACHC were introduced into endomorphins to examine the conformational effects on the bioactivity. Use of a combination of receptor binding techniques, (1)H NMR, and molecular modeling allowed the conclusion that Pro (2) substitution by these residues causes changes in structure, proteolytic stability, and pharmacological activity. It seems that the size of the alicyclic beta-amino acids does not have marked influence on the receptor binding affinities and/or selectivities. Among the new analogues, the cis-(1 S,2 R)ACPC (2) and cis-(1 S,2 R)ACHC (2)-containing derivatives displayed the highest binding potencies and efficacies in receptor binding and ligand-stimulated [ (35)S]GTPgammaS functional experiments. Molecular dynamic simulations and (1)H NMR studies of the cis-ACPC/ACHC-containing analogues revealed that many conformations are accessible, though it is most likely that these peptides bind to the mu-opioid receptor in a compact, folded structure rather than extended.