Radioligand binding techniques were employed to determine the modulation by nucleotides of the specific [3H]glibenclamide (Gli) binding to rat aortic and cardiac ventricular preparations. Saturation analysis revealed a single binding site with K(D) value of 31.3 nM and Bmax of 180 fmol/mg wet weight in aortic preparations. We also observed that [3H]Gli bound reversibly and specifically to cardiac membranes. Unlabeled glibenclamide displaced [3H]Gli-specific binding of cardiac membranes completely with K(I) of 54.4 nM. In cardiac membranes, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and uridine diphosphate (UDP) (from 0.01-5 mM) concentration dependently inhibited [3H]Gli binding independent of Mg2+. The values of K(I) were 0.47, 0.22, and 0.58 mM, respectively. However, in aortic preparations, [3H]Gli-specific binding was increased by ATP of 5 and 10 mM and showed a biphasic response to ADP. At concentrations to 1 mM, ADP inhibited binding; above 5 mM, the specific [3H]Gli binding was increased. UDP did not alter the binding up to 5 mM. In the presence of Mg2+ (20 mM), the inhibitory effects of ATP (0.01-1 mM) or ADP (0.01-5 mM) on the binding in cardiac membranes were abolished, whereas the facilitatory effects of ATP or ADP in aortic preparations were strengthened. Analysis of kinetics showed that the time of [3H]Gli association and dissociation in cardiac and aortic preparations was monophasic. The association was delayed with dissociation unchanged by ATP, ADP, and UDP of 1 mM, respectively, in cardiac membranes. In aorta, however, at the same concentration ATP accelerated association and retarded dissociation and vice versa for ADP. Association and dissociation were not changed by UDP of 5 mM. We conclude that ATP, ADP, and UDP are all major allosteric modulators of K(ATP) channels and they affect the antagonist binding to heart (sulfonylurea receptor 2A) and aorta (sulfonylurea receptor 2B) differently.