Quantitative ligand binding studies resolved two subtypes of the delta opioid receptor, termed delta(ncx1) and delta(ncx2), in mouse brain membranes depleted of mu receptors by pretreatment with the irreversible ligand, BIT. The purpose of the present study was to compare the binding parameters, ligand-selectivity profile and pharmacological properties of the cloned mouse delta receptor (MDOR) stably expressed in a cell line to the delta(ncx) binding sites of mouse brain. [3H][D-Ala2,D-Leu5]enkephalin labeled a single binding site in membranes prepared from MDOR cells under several different assay conditions including BIT-pretreatment. The MDOR had high affinity for delta agonists and antagonists. [3H][D-Ala2,D-Leu5]enkephalin labeled two binding sites in mouse brain membranes depleted of mu receptors by pretreatment with BIT: the delta(ncx1) site (high affinity for DPDPE and deltorphin) and the delta(ncx2) site (low affinity for DPDPE and deltorphin). Some agents were moderately selective for the delta(ncx2) site: [pCl]DPDPE (10.9-fold), JP41 (5.9-fold) and JP45 (3.8-fold). The Ki values of 12 opioids at the mouse MDOR were determined. These values were highly correlated with their values at the delta(ncx1) site but not the delta(ncx2) site. These data suggest that the delta(ncx2) site may be distinct from the cloned delta opioid receptor.