The glycolytic enzyme aldolase is concentrated in a domain around stress fibers in living Swiss 3T3 cells, but the mechanism by which aldolase is localized has not been revealed. We have recently identified a molecular binding site for F-actin on aldolase, and we hypothesized that this specific binding interaction, rather than a nonspecific mechanism, is responsible for localizing aldolasein vivo.In this report, we have used fluorescent analog cytochemistry of a site-directed mutant of aldolase to demonstrate that actin-binding activity localizes this molecule along stress fibers in quiescent cells and behind active ruffles in the leading edge of motile cells. The specific cytoskeletal association of aldolase could play a structural role in cytoplasm, and it may contribute to metabolic regulation, metabolic compartmentation, and/or cell motility. Functional duality may be a widespread feature among cytosolic enzymes.