Associations between glycolytic enzymes and subcellular structures have been interpreted as presenting a novel mechanism of glycolytic control; reversible enzyme binding to subcellular structural components is believed to regulate enzyme activity in vivo through the formation of a multi-enzyme complex. However, three lines of evidence suggest that enzyme binding to cellular structures is not involved in the control of glycolysis. (i) Calculations of the distribution of glycolytic enzymes under the physiological cellular conditions of higher ionic strength and higher enzyme concentrations indicate that a large multi-enzyme complex would not exist. (ii) In many cases, binding to subcellular structures is accompanied by changes in enzyme kinetic parameters brought about by allosteric modification, but these changes often inhibit enzyme activity. (iii) In the case where formation of binary enzyme/enzyme complexes activates enzymes, the overall increase in flux through the enzyme reaction is negligible.