The isolation by affinity chromatography of a low‐molecular‐weight form of phosphofructokinase from Escherichia coli K‐12 has resulted in an improved 2050‐fold purification. The enzyme was readily separable from the higher‐molecular‐weight form by Sepharose 6B fractionation. The enzyme was found to have a molecular weight of 65000 ± 6500 with a subunit molecular weight of 35000 ± 3500. The enzyme exists in either of two interconvertible forms dependent on the presence or absence of the positive of ectors ADP, fructose 6‐phosphate and ATP. A regulatory mechanism is postulated for this enzyme involving hysteric conformational changes; its importance in the mechanism of the Pasteur effect is discussed.