Research into the functional role of the opioid delta receptor has intensified with the recent in vivo identification of delta receptor subtypes, termed delta 1 and delta 2, which mediate antinociception in the mouse. A variety of data also support the hypothesis of an opioid receptor complex composed of distinct, yet interacting, mu, delta, and perhaps kappa binding sites. This model postulates two classes of delta binding sites: a delta binding site not associated with the opioid receptor complex, termed the delta ncx site, and a delta site associated with the receptor complex, termed the delta cx site. A major purpose of this study was to clarify the relationship between the delta ncx binding sites and the delta 1 and delta 2 receptors. Mouse brain membranes were depleted of mu sites by pretreatment with the site-directed acylating agent, BIT, and the delta ncx binding sites were labeled with [3H][D-Ala2,D-Leu5]enkephalin. Binding surface analysis readily resolved two binding sites (delta ncx-1 and delta ncx-2) in the absence and presence of 100 mM NaCl. Control experiments with guanine nucleotides and the ligand-selectivity analysis indicated that the two sites were not two states of a single receptor. Pretreatment of membranes with DALCE, but not [Cys4]deltorphin, decreased [3H] [D-Ala2,D-Leu5]enkephalin and [3H][D-Ser2,Thr6]enkephalin binding. Ligand-selectivity analysis of the two binding sites suggested that neither delta ncx binding site had the characteristics expected of the delta 2 receptor, and that the delta ncx-1 site, but not the delta ncx-2 site, was synonymous with the delta 1 receptor. Moreover, our finding that the racemic nonpeptide delta agonist, BW373U86, had high affinity at and selectivity for the delta ncx-2 site suggests that this site may be a novel delta receptor that mediates some of the effects of BW373U86.