On the basis of evidence that opioid compounds with a mixed μ agonist/δ antagonist profile may produce an antinociceptive effect with low propensity to induce side effects, bifunctional opioid peptides containing the μ agonist H‐Dmt‐d‐Arg‐Phe‐Lys‐NH2([Dmt1]DALDA; Dmt = 2′,6′‐dimethyltyrosine) connected tail‐to‐tail via various α,ω‐diaminoalkyl‐ or diaminocyclohexane linkers to the δ antagonists H‐Tyr‐TicΨ[CH2‐NH]Cha‐Phe‐OH (TICP[Ψ]; Cha = cyclohexylalanine, Tic = 1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid), H‐Dmt‐Tic‐OH or H‐Bcp‐Tic‐OH (Bcp = 4′‐[N‐((4′‐phenyl)phenethyl)carboxamido]phenylalanine) were synthesized and pharmacologically characterizedin vitro. Bifunctional [Dmt1]DALDA→NH‐(CH2)n‐NH←TICP[Ψ] compounds (n = 0–12) showed decreasing μ and δ receptor binding affinities with increasing linker length. As expected, several of the bifunctional peptides were μ agonist/δ antagonists with low nanomolar μ and δ receptor binding affinities. However, compounds with unexpected opioid activity profiles, including a μ partial agonist/δ partial agonist, μ antagonist/δ antagonists and μ agonist/δ agonists, were also identified. These results indicate that the binding affinities and intrinsic efficacies of these bifunctional compounds at both receptors depend on the length and type of the linker connecting the μ and δ components. An important recommendation emerging from this study is that thein vitroactivity profiles of bifunctional compounds containing an agonist and an antagonist component connected via a linker need to be determined prior to their pharmacological evaluationin vivo.