The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was recently identified as a new intracellular binding partner for glucokinase (GK). Therefore, we studied the importance of this interaction for the activity status of GK and glucose metabolism in insulin-producing cells by overexpression of the rat liver and pancreatic islet isoforms of PFK-2/FBPase-2. PFK-2/FBPase-2 overexpression in RINm5F-GK cells significantly increased the GK activity by 78% in cells expressing the islet isoform, by 130% in cells expressing the liver isoform, and by 116% in cells expressing a cAMP-insensitive liver S32A/H258A double mutant isoform. Only in cells overexpressing the wild-type liver PFK-2/FBPase-2 isoform was the increase of GK activity abolished by forskolin, apparently due to the regulatory site for phosphorylation by a cAMP-dependent protein kinase. In cells overexpressing any isoform of the PFK-2/FBPase-2, the increase of the GK enzyme activity was antagonized by treatment with anti-FBPase-2 antibody. Increasing the glucose concentration from 2 to 10 mmol/l had a significant stimulatory effect on the GK activity in RINm5F-GK cells overexpressing any isoform of PFK-2/FBPase-2. The interaction of GK with PFK-2/FBPase-2 takes place at glucose concentrations that are physiologically relevant for the activation of GK and the regulation of glucose-induced insulin secretion. This new mechanism of posttranslational GK regulation may also represent a new site for pharmacotherapeutic intervention in type 2 diabetes treatment.