After exposure of monkey kidney epithelial cells to a reduced concentration of K, a known mitogenic signal, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD) is activated by a cytosolic protein whose function appears to be novel. A monospecific antibody was used as an immunoprobe to study the contribution of this G3PD modifier protein (MP) to signal transduction. Raising the extracellular Na concentration as well as lowering the K concentration of the medium increased the amount of MP in cytosol and also activated G3PD. Metabolic labeling of cells followed by preparation of detergent-soluble (cytosolic) and detergent-resistant (cytoskeletal) fractions, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled immune precipitates suggested that the protein was also associated with cytoskeleton. Depolymerization of the microtubules with colchicine or nocodazole increased cytosolic immunoreactive MP, whereas cytochalasin D had no effect. Taxol, which stabilizes microtubules, blocked the effects of colchicine or nocodazole. When tubulin, actin, and intermediate filament fractions of the cytoskeleton were prepared, blotted, and probed with specific antibodies, MP was found in the tubulin fraction. These observations suggest that MP is associated with the microtubules and can be displaced into the cytosol, wherein it could activate G3PD and thereby stimulate glycolytic production of ATP during mitogenic signal transduction.