BACKGROUND Opioid agonist activation at the mu opioid receptor (MOR) can lead to a wide variety of physiological responses. Many opioid agonists share the ability to selectively and preferentially activate specific signaling pathways, a term called biased agonism. Biased opioid ligands can theoretically induce specific physiological responses and might enable the generation of drugs with improved side effect profiles. METHODS Dynorphins, enkephalins, and endomorphins are endogenous opioid agonist peptides that may possess distinct bias profiles; biased agonism of endogenous peptides could explain the selective roles of these ligands in vivo. Our purpose in the present study was to investigate biased signaling and potential underlying molecular mechanisms of bias using 35S-GTPγS and cAMP assays, specifically focusing on the role of adenylyl cyclases (ACs) and regulators of G-protein signaling proteins (RGSs) in CHO, N2a, and SH-SY5Y cell lines, all expressing the human MOR. RESULTS We found that endomorphin-1/2 preferentially activated cAMP signaling, while dynorphin-B preferentially activated 35S-GTPγS signaling in most cell lines. Experiments carried out in the presence of an isoform selective RGS-4 inhibitor, and siRNA knockdown of AC6 in N2a cells did not significantly affect the bias properties of endomorphins, suggesting that these proteins may not play a role in endomorphin bias. CONCLUSIONS We found that endomorphin-1/2 and dynorphin-B displayed contrasting bias profiles at the MOR, and ruled out potential AC6 and RGS4 mechanisms in this bias. This identified signaling bias could be involved in specifying endogenous peptide roles in vivo, where these peptides have low selectivity between opioid receptor family members.