The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa 1 is the dominant receptor subtype, frog brain contains mainly the kappa 2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa 2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain. Kappa 1 binding sites measured in the presence of 5 microM/D-Ala2-Leu5/enkephalin represent 25-30% of [3H]ethylketocyclazocine binding in frog brain membranes. The kappa 2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.