The role of fructose 2,6-bisphosphate in the regulation of glycolysis in the citric acid accumulating fungus Aspergillus niger was investigated. Fructose 2,6-bisphosphate stimulated the activity of partially purified phosphofructokinase by increasing the affinity of the enzyme for fructose 6-phosphate and relieving inhibition by ATP. Fructose 2,6-bisphosphate acted synergistically with AMP, but not with NH,+ ions, which otherwise also activate phosphofructokinase. Fructose 2,6-bisphosphate also partially antagonized citrate inhibition of phosphofructokinase; complete deinhibition against high (5 mM) concentrations of citrate (as occur during citric acid accumulation), however, required the simultaneous presence of fructose 2,6bisphosphate (0.1 PM), AMP (0.1 mM) and NH,+ ions (20 mM).