Endogenous mu-opioid receptor (MOR) selective peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), unlike so called 'typical opioids', are characterized by the presence of Pro(2) residue, which is a spacer connecting aromatic pharmacophoric residues. In order to investigate structural requirements for position 2, we synthesized endomorphin analogs incorporating, instead of Pro, unnatural amino acids with six-membered heterocyclic rings, such as piperidine 2-, 3- or 4-carboxylic acids (Pip, Nip and Inp, respectively). (R)-Nip residue turned out to be favourable for improving MOR affinity. Introduction of 2',6'-dimethyltyrosine (Dmt) instead of Tyr(1) led to obtaining [Dmt(1), (R)-Nip(2)]EM-2 which showed exceptional MOR affinity and high stability against enzymatic degradation in rat brain homogenate. In in vivo hot-plate test in mice, this analog given intracerebroventicularly (i.c.v.), produced profound supraspinal analgesia, being much more potent than EM-2. The antinociceptive effect of this analog lasted about 170 min and was almost completely reversed by beta-funaltrexamine (beta-FNA), a selective MOR antagonist.