Steady-state analysis of nucleotide-binding sites on rat liver plasma membranes was carried out using 3H-labelled ATP as radioligand under complete inhibition of ecto-ATPase activity by excess EDTA. Binding of [3H]ATP to the membranes is saturable, reversible and apparently involves one population of specific binding sites with Kd of about 90 nM and binding capacity (Bmax) of 15 pmol/mg protein. A broad spectrum of purinergic agonists and antagonists was examined as potential inhibitors of the measured binding. The displacement studies showed the following rank order of inhibitory potency for [3H]ATP-binding sites (pIC50 values in parentheses): ATPgammaS (7.49)>2-MeSATP (7.18)>ATP (6.91)>ADPbetaS (6.64)>/=ADP (6.56)>>RB2 (6.14)>>suramin (5.40)>>Ap4A (4. 57)>alpha,beta-MeATP (4.19)>/=beta,gamma-MeATP (3.97). AMP, adenosine, Ap5A, PPADS, beta-glycerophosphate as well as non-adenine nucleoside triphosphates GTP, UTP and CTP did not exert any effect on the measured binding at concentration ranges of 10-6-10-4 M. In order to ascertain whether ATP and its analogues are capable of interacting with the same binding domain, 2-MeSATP and ADP were treated as alternative ligands that could compete with unlabelled ATP for its binding sites. A 2-fold increase of Kd value for ATP-receptor interaction was observed in the presence of 2-MeSATP (60 nM) or ADP (250 nM) without any modulation of Bmax value, confirming that inhibitory effects of these compounds are competitive in nature. These studies demonstrate that ATP and its analogues are able to interact with a single binding domain on liver plasma membranes, which may be identified as ligand-binding component of P2 purinoceptors of the P2Y1 subtype.