F-protein, a consistent contaminant of myosin preparations, has been shown to be phosphofructokinase, the key regulatory enzyme of glycolysis. In homogenates of rigor muscle most of the phosphofructokinase sediments with the myofibrils, suggesting that in the living muscle cell phosphofructokinase is not in the soluble fraction as was formerly thought, but bound to the myofibrils. Fluorescent antibody to F-protein labels myofibrils in a zone in each half of the A-band. The increase in separation of the zones across the A-band with increase in sarcomere length suggests that the antibody binds to the parts of the cross-bridge regions of the thick filament within the H-zone. It therefore seems likely that phosphofructokinase is located in the cross-bridge region of the thick filament, but that access of antibody is restricted by overlapping thin filaments.