Our laboratory was among the first to propose the existence of delta receptor subtypes: a delta site thought to be associated with a mu-delta-opioid receptor complex termed the delta cx binding site and delta site not associated with the mu-delta-opioid receptor complex, termed the delta ncx site. In previous studies, we assayed the delta cx site with [3H][D-Ala2,D-Leu5]enkephalin using rat brain membranes depleted of delta ncx sites by pretreatment with the site-directed acylating agent, (+)-trans-SUPERFIT. In the present study, we investigated, using (+)-trans-SUPERFIT-pretreated membranes, the possibility of heterogeneity of the delta cx binding site. Two sites were resolved: the delta cx-1 site at which mu ligands are potent noncompetitive inhibitors and delta ligands are weak competitive inhibitors, and the delta cx-2 site where delta ligands are potent and mu ligands are weak, mixed competitive-noncompetitive inhibitors. Although the delta cx-2 site has a delta-like ligand-selectivity profile, several experiments distinguished it from the delta ncx site. Two lines of evidence suggest that the delta ncx site corresponds to the cloned delta receptor. One, the delta receptor was cloned from the NG108-15 cell line, and this receptor, like the delta ncx binding site, irreversibly binds SUPERFIT and (+)-trans-SUPERFIT. Secondly, administration of delta-antisense DNA selectively decreases delta ncx binding. Viewed collectively, the major finding of this study is the discovery of a novel SUPERFIT-insensitive and delta-antisense-insensitive delta cx-2 binding site.