6-phosphofructo-1-kinase (PFK) is a key regulatory enzyme of the glycolytic flux through the Embdem- Meyerhoff pathway. This enzyme is under a tight regulation and its activity is modulated by several physiological ligands, such as ATP, citrate, fructose-2,6-bisphosphate, AMP and cyclic-AMP. PFK undergoes several oligomeric conformers, which dimmers are inactive and tetramers, as well as more complex conformers, are totally active. The equilibrium between the oligomers is regulated by PFK effectors and is possible one of the mechanisms of the enzyme regulation under physiological conditions. Here we studied the effects of the chaotropic agent, guanidinium hydrochloride (Gnd-HCl), on the activity and the oligomeric equilibrium of PFK, as a model to evaluate the relationship between PFK activity and oligomeric configuration. Gnd-HCl inhibits PFK in a dose-dependent manner, presenting a Ki of 0.23 ± 0.02 M and a cooperativity index (n) of 3.3 ± 0.3 observed in the presence of 5 µg/ml PFK. These parameters significantly augmented to 0.32 ± 0.03 M (Ki) and 4.1 ± 0.4 (n) in the presence of 10 µg/ml PFK (P < 0.05, compared to 5 µg/ml PFK, Student's t-test), and 0.41 ± 0.04 M (Ki) and 6.2 ± 0.6 (n) in the presence of 20 µg/ml PFK (P < 0.05, compared to 5 or 10 µg/ml PFK, Student's t-test). These data suggest that the enzyme becomes more resistant to Gnd-HCl in a function of increasing enzyme concentrations. Enzyme titration experiments revealed that the affinity constant to conversion of dimmers in tetramers increased from 5.3 ± 0.5 nM to 338 ± 32 nM in the presence of 0.5 M Gnd-HCl (P < 0.05, Student's t-test). The allosteric ligands ATP and fructose-2,6-bisphosphate increased the resistance of PFK to Gnd-HCl in a significative way, increasing the Ki for inactivation. These data suggest that Gnd-HCl is shifting the enzyme to a less complex oligomeric configuration (monomers and dimmers) that inhibit the enzyme, and that the denaturation of PFK occurs through formation of monomers. Additionally, data presented here support evidences that ATP and fructose-2,6-bisphosphate exert their effects shifting the oligomeric equilibrium of the enzyme.