In solubilized extracts from bovine striatal membranes three different binding sites for 5'-N-ethylcarboxamidoadenosine ([3H]NECA) were observed after separation of the extract by gel filtration on Sepharose CL-6B. The first peak was eluted in the void volume and contained the A2 adenosine receptor. In the second peak, [3H]NECA binding sites were eluted with a pharmacological profile characteristic of adenotin, a low affinity non-receptor adenosine binding protein. The third peak represented approximately 50% of the [3H]NECA binding activity. This site bound [3H]NECA in a reversible and saturable manner with KD of 17 nmol/l and a binding capacity of 11.3 pmol/mg protein. In competition experiments, adenosine, NECA, NAD, inosine, 5'-AMP and S-adenosyl-L-homocysteine were the most potent ligands. In contrast to adenosine receptors, this site did neither bind adenosine receptor antagonists nor the A2 selective agonist CGS 21,680 (2-[p-(2-carboxyethyl)phenethylamino]5'-N-ethylcarboxamidoadeno sin e). These results suggest the existence of a novel high affinity binding site for adenosine of unknown function in bovine striatum.