A series of five epimeric pairs of naltrexone derivatives that contain an electrophilic substituent at the 6a- or 6β-position was synthesized and tested on the guinea pig ileal longitudinal muscle (GPI) and mouse vas deferens (MVD) preparations in order to determine if the orientation of the electrophile is important for covalent bonding to opioid receptors. In the GPI all compounds were pharmacologically active as reversible agonists, but only the 6/3-isomers of the fumaramate ester 2b (β-FNA) and isothiocyanate 6b exhibited covalent reactivity, involving a selective irreversible antagonism of the μ agonist, morphine, without affecting k agonists. The 6a-isomer 2a (α-FNA) was itself nonalkylating but was able to protect the GPI against alkylation by its epimer, β-FNA, indicating that the two epimers bind to the same receptor. These results suggest that the proper orientation of the electrophilic substituent is required for covalent bonding with a proximal nucleophile in the case of μ receptor blockade. Moreover, the lack of covalent bonding to k receptors by these or other ligands in this series indicates the possible absence of sufficiently reactive nucleophiles on this recognition site. In the MVD, 2b, but not 2a, irreversibly antagonized morphine (as in GPI), whereas neither epimer exhibited irreversible antagonism toward the δ agonist, [d-Ala2,D-Leu5]enkephalin (DADLE). In contrast, both of the isothiocyanate epimers (6a,b) irreversibly blocked μ and & receptors. Evidence suggesting differences between μ receptors in the MVD and GPI was obtained with the /3-iodoacetamide 5b, which was an irreversible blocker of morphine only in the MVD. When analyzed together with those of previous studies with the nitrogen mustard analogues, a- and /3-chlornaltrexamine, the data suggest that the receptor-alkylating ability of each isomer in an epimeric pair differs most when the electrophile possesses a narrow spectrum of reactivity.