The effect of polyethylene glycol (PEG) on lactate production from glucose by rat liver postmitochondrial fraction has been examined, using physiological concentrations of substrate and effectors. Addition of 10% polyethylene glycol (Mw 6000) resulted in an increase in lactate production, while the inhibition of lactate production by citrate and ATP was decreased. These results in conjunction with analysis of glycolytic intermediates suggest that PEG exerts its major action on phosphofructokinase.