The modulation of adenosine receptor with K+ATPchannel blocker, glibenclamide, was investigated using the radiolabeled A2A-receptor selective agonist [3H]CGS 21680. Radioligand binding studies in bovine brain striatal membranes (BBM) indicated that unlabeled CGS 21680 displaced the bound [3H]CGS 21680 in a concentration-dependent manner with a maximum displacement being approximately 65% at 10−4m. In the presence of 10−5m glibenclamide, unlabeled CGS 21680 increased the displacement of bound [3H]CGS 21860 by approximately 28% at 10−4m. [3H]CGS 21680 bound to BBM in a saturable manner to a single binding site (Kd=10.6±1.71n m; Bmax=221.4±6.43fmol/mg of protein). In contrast, [3H]CGS 21680 showed saturable binding to two sites in the presence of 10−5m glibenclamide; (Kd=1.3± 0.22n m; Bmax=74.3±2.14fmol/mg protein; and Kd=8.9±0.64n m; Bmax=243.2±5.71fmol/mg protein), indicating modulation of adenosine A2Areceptors by glibenclamide. These studies suggest that the K+ATPchannel blocker, glibenclamide, modulated the adenosine A2Areceptor in such a manner that [3H]CGS 21680 alone recognizes a single affinity adenosine receptor, but that in the presence of glibenclamide it binds to two sites. These studies provide further insight into the interactions between K+ATPchannels and adenosine receptors.