The crude membrane fraction from the brain of the frog Rana ridibunda was shown to contain 0.7-0.8 pmol/mg protein for a site with high (KD = 0.1 nM) and about 3.2 pmol/mg protein for a site with lower (KD = 10-15 nM) affinity for the opiate agonist [3H]etorphine and for the opiate antagonist [3H]diprenorphine. In addition to its very high affinity for the two tritiated oripavine derivatives, the high affinity site displayed (i) a considerably reduced ability to bind the agonist but not the antagonist in the presence of Na+ ions and (ii) pronounced stereospecificity. These properties are all typical of an opioid receptor site. The lower affinity site, which was about four times as abundant as the other exhibited none of the aforementioned characteristics and is therefore probably not opioid in nature. Detailed testing of the potency of various unlabelled opioid ligands to inhibit the binding of [3H]etorphine at the high affinity site showed that the latter consists of a mixture of several types of opioid sites, including a major type with an apparent binding profile clearly different from those of mammalian brain mu, delta- and kappa-opioid sites. In particular, this major type of site, which accounted for about 70% of the opioid binding in frog brain membranes, bound mu ([D-Ala2,MePhe4,Glyol5]enkephalin), delta ([D-Thr2,Leu5]enkephalyl-Thr) and kappa (U50,488) selective ligands with much lower affinity than did mu-, delta- and kappa-opioid receptor sites, respectively.