Biochemical identification of mitochondrial sulfonylurea receptors has been carried out through binding studies performed with [3H]glibenclamide. The presence of a single class of low affinity binding sites for glibenclamide in the inner mitochondrial membrane has been found, with a KD of 360 +/- 48 nM and BMAX of 48 +/- 7 pmoles/mg in beef heart mitochondria. Glibenclamide binding was affected by other sulfonylureas (glipizide, glisoxepide) but not by potassium channel openers (diazoxide, pinacidil, RP66471). In both rat liver and beef heart mitochondria adenine nucleotides (ATP, ADP, AMP) and nucleotide analogs (triazine dyes) produced large inhibition (from 60 to 80%) of [3H]glibenclamide binding. Photoaffinity labeling of submitochondrial particles with [125I]-glibenclamide revealed a single specifically labeled polypeptide band of 28 kDa by SDS-PAGE that is postulated to be (or to form a part of) the mitochondrial sulfonylurea receptor.