A radioligand binding assay was carried out to explore the key structure in molecules of ATP and its analogues responsible for the binding to P2x-purinoceptors. 2. It was found that adenosine, adenine and xanthine had no significant effect on [3H]alpha, beta-methylene ATP binding to membrane fractions prepared from rat urinary bladder, while pentasodium triphosphate and disodium pyrophosphate could effectively displace the binding. Sodium orthophosphate was shown to displace the binding only at much higher concentration. 3. Apart from ATP, several other nucleotides could also fully displace the specific binding, but with potencies lower than that of ATP. 4. The results indicate that the phosphate side chain in molecules of ATP and its analogues is the key structure responsible for the binding to P2x-purinoceptors.