The modification of pyruvate kinase activity in vitro was examined by altering the environmental [Mg(2+)]/[Ca(2+)] ratio with EDTA on the one hand and isolated rat liver mitochondria on the other. 2. Controlled additions of Ca(2+) and EDTA caused pyruvate kinase activity to be alternately and rapidly switched on and off. 3. By being able to accumulate Ca(2+) in preference to Mg(2+) rat liver mitochondria were able to alter the [Mg(2+)]/[Ca(2+)] ratio in the vicinity of pyruvate kinase and thereby modify the activity of this enzyme. 4. The possible role of mitochondria in modifying pyruvate kinase and other ion-sensitive cytoplasmic enzyme activities is discussed.