Fructose 1,6-bisphosphate aldolase inactivation by L- and D-glyceraldehyde 3-phosphate (Ga 3-P) obeys pseudo first-order kinetics. L-Ga 3-P is much more effective than the D-isomer: the Ki values obtained are 0.032 mM and 0.54 mM respectively. Kinetic analysis suggests that one residue of the active center region is involved in the inactivation mechanism: specifically, a cysteine residue appears to be responsible for the initial inactivation by L-Ga 3-P. Lysine and arginine residues become involved at further steps of the inactivation mechanism. No correlation between loss of thiol groups and decay of catalytic activity was observed for the enzyme treated with D-Ga 3-P. The role of lysine and arginine residues in this reaction is discussed.