1. The effects of the Mg complex of adenosine 5'-triphosphate (MgATP) on binding of sulphonylureas to microsomes obtained from mouse pancreatic islets were examined. 2. MgATP inhibited the binding of both glibenclamide and tolbutamide to microsomes. 3. Binding of [3H]-glibenclamide inhibited by MgATP was not further diminished by Mg(2+)-bound adenosine 5'-(beta, gamma-imidotriphosphate) (AMP-PNP) or free adenosine 5'-diphosphate (ADP). Higher concentrations of MgAMP-PNP induced a partial reversal of the inhibitory effect of MgATP on [3H]-glibenclamide binding. 4. The apparent dissociation constant (K'D) for binding of [3H]- glibenclamide remained constant when 5. Extracellular ADP did not markedly stimulate insulin release from mouse pancreatic islets. 6. It is concluded that sulphonylureas and cytosolic nucleotides exert their inhibitory effects on the K-ATP-channels of beta-cells by binding to different sites. The binding properties of the sulphonylurea receptor seem to be modulated by protein phosphorylation.