Phosphofructokinase activity was determined in extracts from ossicles of otosclerotic and non-otosclerotic individuals. The assay was varied to determine the influence of adenosine triphosphate and citrate on bone phosphofructokinase activity, especially in otosclerotic stapedes. Phosphofructokinase from ossicles is not inhibited by levels of adenosine triphosphate which normally inhibit the enzyme from skeletal muscle, heart, liver and brain tissue. The phosphofructokinase activity in stapedes is greater than the other ossicles, but the activity from otosclerotic stapedes is similar to the activity from non-otosclerotic controls. However, otosclerotic stapedes phosphofructokinase responds differently and with less sensitivity to increasing citrate concentrations than non-otosclerotic controls. Glucose-6-phosphate, replacing fructose-6-phosphate in the phosphofructokinase reaction mixture, yields rates similar to fructose-6-phosphate, indicating hexose isomerase is functioning to the same capacity in otos...