The non-P1 and non-P2 muscle relaxant effect of ATP in rabbit thoracic aorta has recently been attributed to a putative P3 purinoceptor, which is activated by either adenosine or ATP. Since the physiological roles of this putative P3 purinoceptor and of a new [3H]-5‘-N-ethylcarboxamidoadenosine (NECA)-binding protein from rat brain membranes called P3 purinoceptor-like protein (P3LP), due to its ligand specificity, have not been fully elucidated, we needed a specific ligand to obtain further information about the receptor. We examined the structure−activity relationship (SAR) of various 5‘-N-substituted-carboxamidoadenosine derivatives toward P3LP and discovered a hydrophobic binding region near the 5‘-N-substituted-carboxamide group. From the linear alkyl N-substituted derivatives, the N-n-pentyl derivative 10 was found to be the most potent ligand with a Ki value of 12 nM. In the series of the N-cycloalkyl derivatives, the N-cyclohexyl derivative 27 was the strongest ligand with a Ki value of 18 nM. On the other hand, the N-substituents having branched alkyl side chains and bulky cycloalkyl groups did not show any potent affinities for P3LP. Therefore, the hydrophobic pocket accommodates approximately a 10-carbon-atom-long linear alkyl side chain, while a considerably stronger hydrophobic binding region of about a 5-carbon-atom-long depth exists near the nitrogen atom of the amide group. This pocket also allows substitution with bulky hydrophobic groups since the 5‘-N-cycloalkyl derivatives have high affinities. We also examined the receptor selectivity for the selected nucleosides 10 and 27 with 1 [9-(6,7-dideoxy-β-d-allo-hept-5-ynofuranosyl)adenine, HAK2701] and NECA versus P1 purinoceptor subtypes, such as adenosine A1, A2A, A2B, and A3 receptors, and found that 27 is the most selective ligand for P3LP.