We now report on another line of evidence which provides additional support for the multiple modality or multiple receptor concept. Through use of the novel pair of receptor probes, 1 (β-FNA) and 2 (β-FOA), we present data which suggest that only 1 covalently binds to opioid receptors even though each of the compounds contains an identical alkylating moiety and forms a receptor complex. β-FNA (1) behaved as a potent reversible agonist with an IC50 of 4.8 × 10^-9 M when tested on the electrically stimulated guinea pig ileal longitudinal muscle preparation. The agonist effect of 1 could be reversed at all incubation times by washing or addition of naltrexone (7). However, when the ileum was incubated with 1 (2 × 10^-7 M) for different time periods and then washed (20X), a time-dependent, irreversible narcotic antagonistic effect against morphine was observed. The specificity of the blockage was suggested by the fact that the irreversible effects of 1 could be inhibited by prior addition of the reversible antagonist, naltrexone (7; 5 × 10^-6 M). Norepinephrine receptors were unaffected by β-FNA treatment. In view of the generally known chemical properties of Michael acceptors, the SH group is a good candidate for the nucleophile which forms a covalent bond with β-FNA (1). Significantly, when the ileum was treated with 2 (β-FOA) under conditions identical with those for 1, it produced a reversible agonistic effect (IC50 = 2.7 × 10^-8 M) but no agonism or morphine antagonism was observed following washing (20X) after 80 min of incubation. The agonistic effect of 2 also was blocked by the irreversible action of 1. Since the data suggest that 1 but not 2 forms a covalent bond with opioid receptors, it is likely that the receptor environments which interact with the fumaramate ester moiety of 1 and 2 are different. This indicates either that 1 and 2 interact differently with a single receptor or that they associate with different receptors, as proposed in the original concept.