Opioid agonists produce analgesia in mammals through the activation of µ, κ, or δ opioid receptors. Previous behavioral and binding studies from our laboratory using an amphibian model suggested that, µ, κ, or δ opioid agonists may activate a single type of opioid receptor in the grass frog, Rana pipiens. In the present study, kinetic, saturation, and competitive binding profiles for three opioid radioligands, [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) (µ-selective), [3H]U65953 [(5α,7α,8β)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide] (κ-selective), and [3H]DPDPE ([d-Pen2,d-Pen5]-enkephalin) (δ-selective) were determined using frog whole brain homogenates. Kinetic analyses and experimentally derived values from saturation experiments gave affinity constants (KD) in the low nanomolar range. The density of opioid binding sites (Bmax) was 224.4, 118.6, and 268.9 fmol/mg for µ, κ, and δ opioid radioligands, respectively. The affinity values did not significantly differ among the three opioid radioligands, but the κ radioligand bound to significantly fewer sites than did the µ or δ radioligands. Ki values for unlabeled µ, κ, and δ competitors, including highly selective opioid antagonists, were consistent with each radioligand selectivity profile. The present data suggest that µ, κ, and δ opioid radioligands bind to distinct opioid receptors in amphibians that are surprisingly similar to those found in mammalian brain.