Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose-6-phosphate (Fru-6-P) by MgATP in the first committed step of glycolysis. Bacterial phosphofructokinase is an allosteric, homotetrameric enzyme and is allosterically regulated by phospho(enol)pyruvate (PEP) and MgADP. In phosphofructokinase from both Bacillus stearothermophilus (BsPFK) and Escherichia coli (EcPFK) the allosteric coupling between PEP and Fru-6-P results from compensating enthalpy and entropy components. In EcPFK the positive coupling free energy associated with inhibition by PEP is established by the positive enthalpy component. In BsPFK the sign of the coupling free energy is opposite that of the enthalpy term and is therefore determined by the larger absolute value of the entropy term. This striking contrast between BsPFK and EcPFK suggests that fundamentally different mechanisms may be responsible for transmitting the allosteric signal between binding sites. In the present study, enzyme activity and allosteric couplings were determined at varying concentrations of urea for both BsPFK and EcPFK. The specific activity of BsPFK increases at low concentrations of urea, reaching 115% of initial activity at ∼0.5M urea. The activity remains at this level until ∼2.5M urea, where it decreases until reaching 0% at ∼6M urea. EcPFK slowly loses activity from 0 to ∼1.5M urea, where the activity begins to quickly decline and is completely lost by ∼3M. Compared to 0M conditions, the allosteric coupling between PEP and Fru-6-P increases by about 3-fold in 0.5M urea, 2-fold in 2M urea, and decreases by 2-fold in 4M urea. This is in direct contrast to EcPFK, where the allosteric coupling is similar at 0, 0.5M and 2M urea. These results support the notion of different underlying mechanisms for inhibition of PFK in Bacillus stearothermophilus and Escherichia coli. Funding: NIH-GM33261, NIH-CBI, Welch-A1543.