Covalent labeling of nucleotide binding sites of the purified sulfonylurea receptor has been carried out with ~y-~~P-labeolexidd ized ATP. The main part of 32Pi ncorporation is in the 145-kDa glycoprotein that has been previously shown to be the sulfonylurea binding protein (Bernardi et al., 1988). ATP and ADP protect against this covalent labeling with values of 100 pM and 500 pM, respectively. Nonhydrolyzable analogs of ATP also inhibit 32P incorporation. Interactions between nucleotide binding sites and sulfonylurea binding sites have then been observed. AMP-PNP, a nonhydrolyzable analog of ATP, produces a small inhibition of [3H]glibenclamide binding (20-296) which was not influenced by Mg2+. Conversely, ADP, which also produced a small inhibition (20%) in the absence of Mg2+, produced a large inhibition (-80%) in the presence of Mg2+. This inhibitory effect of the ADP-Mg2+ complex was observed with a value of 100 f 40 pM. All the results taken together indicate that ATP and ADP-Mg2+ binding sites that control the activity of KATP channels are both present on the same subunit that bears the receptors for antidiabetic sulfonylureas.