We aimed at comparing the binding characteristics of adenosine A(1) and A(2A) receptors (A(1)Rs and A(2A)Rs) in high-expressing cerebral areas, the cortex and striatum respectively, of human, bovine and rat brain. Adenosine A(3) receptor (A(3)R) binding was studied in rat and bovine testis. Results confirmed species differences in AR saturation-displacement binding parameters. To investigate A(3)Rs in CNS, we carried out immunoblot in human brain, resolving two signals, a 52 KDa band with the highest density in hippocampus and a 48 KDa one, slightly more expressed in cortex. Subsequently, A(3)R binding was performed by [I-125]-4-aminobenzyl-5'-N-methylcarboxamidoadenosine ([I-125]-AB-MECA) in human hippocampus, revealing an high affinity population of sites and another non saturable component. [I-125]-AB-MECA first site displacement by N-6 (3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and 1,3-dipropyl-8-cyclopenthyl-xanthine (DPCPX) distinguished two affinity sites, being only in part identified as A(3)Rs. Therefore, A(3)Rs result clearly expressed by Western blot in human brain, but their full CNS characterization needs further investigation.