Approximately 23% of the glycolytic enzyme aldolase in the perinuclear region of Swiss 3T3 cells is immobile as measured by FRAP. Previous studies suggest that the immobile fraction may be associated with the actin cytoskeleton (Pagliaro, L. and D. L. Taylor. 1988. J. Cell Biol. 107:981-991), and it has been proposed that the association of some glycolytic enzymes with the cytoskeleton could have functional significance, perhaps involving a fundamental relationship between glycolysis, cytoplasmic organization, and cell motility. We have tested the effect of a key glycolytic inhibitor and an actin cytoskeletal modulator on the mobility of aldolase in living cells directly, using fluorescent analog cytochemistry and FRAP. We report here that the competitive hexokinase inhibitor 2-deoxyglucose releases the bound fraction of aldolase in 3T3 cells within 10 min, and that this process is reversible upon washout of the inhibitor. A similar result is produced with the actin-binding agent, cytochalasin D. These results are consistent with models in which glycolytic enzymes are not exclusively diffusion-limited, soluble proteins, but may exist partially in the solid phase of cytoplasm. Such organization has significant implications for both the modulation of cytoplasmic structure and for cellular metabolism.