The binding of hexo-/glucokinase and glycerol kinase to mitochondria via the channel forming protein, porin, in pancreatic islet β-cells and adipocytes, was recently proposed to participate in nutritional signaling, glucose sensing, and the control of high-energy phosphate distribution and oxidative phosphorylation. In this study we demonstrate that polyclonal antisera against purified rat liver porin recognize unique proteins in rat pancreatic islets, adipocytes, and RINm5F cells, each with an apparent Mr about 2000 smaller than that of liver porin. Immunoblotting of subcellular fractions, the purity of which has been controlled by the distribution of marker proteins, revealed the mitochondrial localization of the cross-reacting proteins. Their enrichment with a method used for the purification of porin proteins, the characteristic behavior during isoelectric focusing, and the specific binding of rat liver hexokinase and glycerol kinase to phospholipid vesicles containing the purified cross-reacting β-cell or adipocyte proteins strongly suggest their identity with mitochondrial porin. The subtle differences in the apparent Mr and charge heterogeneity raise the possibility of the existence of porin isoforms expressed in a tissue-specific manner. Anti-porin antisera coimmunoprecipitated hexo-/glucokinase from rat insulinoma cell (RINm5F) and adipocyte mitochondria as determined by subsequent immunoblotting of the immunoprecipitates with polyclonal antisera against yeast hexokinase and rat liver glucokinase, respectively. This indicates that some rat pancreatic glucokinase (54 kDa) and liver hexokinase (102 kDa), respectively, is bound to mitochondrial porin. The major portion of the bound fraction is released from mitochondria after treatment with glucose 6-phosphate. Incubation of RINm5F and fat cells with the insulin releasing sulfonylurea drug, glimepiride (20 nM and 5 μM, respectively) for 30 min reduces the amount of hexo-/glucokinase associated with mitochondria and porin to about 50-30%. The reduced kinase binding activity of porin is preserved after isolation of porin from glimepiride-treated cells, reconstitution into phospholipid vesicles and assaying for glucose 6-phosphate inhibitable binding of rat liver hexokinase. The sulfonylurea tolbutamide (20 μM and 5 mM) is significantly less effective. The sulfonylurea-induced inhibition of hexo-/glucokinase binding to mitochondrial porin does not require glucose metabolism or Ca2+ influx into the cells. These data suggest that the sulfonylurea glimepiride, which is thought to inhibit the ATP-regulated K+-channel in β-cells, may have, in addition, an intracellular site of action in pancreatic islet and adipocyte cells at the level of regulation of gluco-/hexokinase binding to mitochondrial porin.