An aspartic acid at residue 95 (Asp95) in the delta receptor has previously been shown to be critical for the binding affinity of selective delta agonists. To gain a better understanding of the functional consequence of agonist action at the delta receptor, the Asp95 residue was mutated to an asparagine (D95N) and opioids were tested for binding and functional activation of the wild-type and mutant delta receptors. Selective agonists such as [D-Ser2,D-Leu5]enkephalin-Thr6 (DSLET) and [D-Ala2,D-Leu5]enkephalin (DADLE) had greatly reduced affinity for the D95N mutant receptor but still inhibited cAMP accumulation, which indicated that the mutant receptor was still functionally coupled to adenylyl cyclase. Antagonist binding was not affected by the Asp95 mutation. Similarly, the partial agonist buprenorphine bound with equally high affinity to the D95N mutant and the wild-type delta receptor, which indicated that Asp95 is not essential for the binding affinity of this opioid. Buprenorphine did not affect cAMP accumulation in HEK 293 cells expressing the D95N mutant, and it blocked the ability of DSLET and bremazocine to inhibit cAMP accumulation via the D95N mutant, which indicated that buprenorphine acts as an antagonist at the D95N mutant. These findings confirm the essential role of Asp95 in the activation of the delta receptor by agonists and reveal a molecular basis of the unique property of buprenorphine.