To clarify the role of glucose-1,6-P2, fructose-2,6-P2 and fructose-1,6-P2 in the control of the glycolytic flux during muscle contraction, we have determined the activity of muscle phosphofructokinase in the presence of physiological concentrations of these bisphosphorylated hexoses and other allosteric effectors, and at increasing pH values. In the presence of fructose2, 6-P2, both glucose-1, 6-P2 and fructose-1,6-P2 can additionally activate the enzyme and partially counteract citrate inhibition. Activation of phosphofructokinase produced by alkalinization increases in the presence of the bisphosphorylated hexoses. It is suggested that the hexose bisphosphates could play a significant role in the initial burst of the glycolytic flux during muscle contraction, when an alkaline pH shift is produced. © 1990 Academic Press, Inc.