Liver phosphofructokinase can be selectively precipitated by the addition of protamine sulfate to a heat-treated crude extract and redissolved, giving nearly full recovery of catalytic activity in combination with a 67-fold increase in specific activity. We have incorporated protamine sulfate precipitation into a five step purification procedure that can be completed in one day--giving 47% recovery of electrophoretically homogeneous liver phosphofructokinase having a specific activity of 50 units/mg. The radio-labeled fragment isolated from a CNBr digest of liver phosphofructokinase that has undergone in vitro phosphorylation catalyzed by the cAMP-dependent protein kinase has the sequence AEYVSGELEHVTRRSLS.