Affinity of ligands for delta opioid receptors (DORs) is regulated by concentration of divalent cations, whereas affinity for alpha2 adrenoceptors is not. In intact NG108‐15 cells, we have demonstrated a 4‐fold increase in affinity of DOR ligands by stimulation of 5‐HT3 receptors, a ligand‐gated cation channel. However, whether this regulation of affinity translates to a regulation of function has not been determined. Accordingly, we assessed the effect of the calcium/magnesium ionphore, A23187 and the 5‐HT3 receptor agonist, 2‐methyl‐5‐HT on delta opioid receptor cell signaling. In intact NG108‐15 cells, the effect of DPDPE (0.1nM – 30 nM) or clonidine (1nM ‐ 1μM) on forskolin‐stimulated cAMP production was determined in the absence or presence of A23187 or 3μM 2‐methyl‐5‐HT. As expected, DPDPE inhibited cAMP production dose‐dependently.2M5HT (3μM) produced a biphasic shift in the concentration‐response curve for DPDPE. At low concentrations of DPDPE, 2M5HT shifted the curve 10‐fold leftward, whereas at high concentrations of DPDPE, the curve was not shifted. This effect was attenuated by the 5‐HT3 antagonist, tropisetron. In contrast, 2‐methyl‐5‐HT had no effect on inhibition of forskolin‐stimulated cAMP produced by the alpha2 adrenoceptor agonist, clonidine. These results are evidence that the increase in affinity produced by cation influx translates to a functional increase in potency of agonists for delta opioid receptors. Supported by the Dept. of Pharmacology, LSUHSC.