The influence of polyethylene glycols on the kinetics of purified rat liver phosphofructokinase was examined because polyethylene glycol often has an associative influence on proteins without specifically interacting with them. Polyethylene glycol was found to prevent or slow the spontaneous inactivation, due to subunit dissociation, of rat liver phosphofructokinase which occurs at low protein concentration (10 microgram/ml) in an isotonic, pH 7 buffer in the absence of substrates. This protection is favored by increasing size and concentration of polyethylene glycol. Inclusion of polyethylene glycol in kinetic assays of rat liver phosphofructokinase had little effect on maximal velocity determined under optimal conditions at pH 8. However, the Km value for fructose 6-phosphate measured at pH 7 and 3 mM MgATP was dramatically lowered with increasing polyethylene glycol concentration. The results support the proposal that more aggregated forms of rat liver phosphofructokinase have a decreased Km value for fructose 6-phosphate when assayed with inhibiting concentrations of MgATP.