A series of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) analogues, containing non-cyclic amino acids (Ala, D-Ala, beta-Ala, NMeAla, D-NMeAla or Sar) instead of Pro in position 2 was synthesized, where NMeAla = N-methylalanine and Sar = N-methylglycine, sarcosine. The opioid activity profiles of these peptides were determined in mu and delta opioid receptor (MOR and DOR)-representative binding assays and bioassays in vitro, as well as in the mouse hot-plate test in vivo. Finally, the degradation rates of all analogues in the presence of either rat brain homogenate or selected proteolytic enzymes were determined. Analogues of EM-2 were generally more potent than the respective analogues of EM-1. EM-2 analogues with D-Ala or D-NMeAla were about twofold more potent than the parent peptide and were least prone to degradation by brain homogenate, dipeptydyl peptidase IV and aminopeptidase M. In the in vivo test, [D-Ala(2)]EM-2 and [D-NMeAla(2)]EM-2 showed much higher analgesic potency than EM-2 which confirmed the usefulness of structural modifications in obtaining new leads for pain-relief therapeutics.