Endomorphins have been shown to produce potent analgesia in various rodent models of pain. However, their central administration led to the development of tolerance and physical dependence. Conjugation of C-terminal substance P (SP) fragments to opioids and opioid peptides was previously shown to produce hybrid peptides with strong analgesic activity, with low or no propensity to develop tolerance. In this study, four peptides (2-5) comprised of endomorphin-1 (1) and C-terminal fragments of SP (four or five amino acids, SP(8-11) (2) or SP(7-11) (4), respectively), with an overlapping Phe residue, were synthesized. To overcome low metabolic stability and poor membrane permeability of the peptide, the N-terminus of 2 and 4 was further modified with a C10-carbon lipoamino acid (C10LAA) achieving 3 and 5, respectively. LAA-modification of the hybrid peptides resulted in a significant increase in metabolic stability and membrane permeability compared to peptides 1, 2 and 4. Compound 5 showed potent μ-opioid receptor binding affinity (K(iμ)=3.87 ± 0.51 nM) with dose-dependent agonist activity in the nanomolar range (IC(50)=45 ± 13 nM). In silico modeling was used to investigate the binding modes and affinities of compounds 1-5 in the active site of μ-opioid receptors. The docking scores were in agreement with the K(iμ) values obtained in the receptor binding affinity studies. The more active LAA-modified hybrid peptide showed a lower total interaction energy and higher negative value of MolDock score.