1. Introduction Liver pyruvate kinase can be phosphorylated and simultaneously inactivated by CAMP-dependent pro- tein kinase; the inactivated enzyme can be reactivated by a multifunctional protein phosphatase (review [ 13). The inactivation of pyruvate kinase has been observed in isolated hepatocytes incubated in the presence of glucagon and in this case the decrease in pyruvate kinase activity was correlated with the stimulation of gluconeogenesis [2]. The glucagon induced inactiva- tion of pyruvate kinase is however transient and reac- tivation occurred within 20 min when sub-optimal doses of glucagon were added. This reactivation was more rapid in the presence of insulin [2,3]. The purpose of this work was to investigate the nature of the enzyme that reactivates pyruvate kinase and its relationship with the well-known protein plios- phatases that act on glycogen phosphorylase a. 2. Methods The inactive form of pyruvate kinase was partially purified from livers of anesthetized rats given glucagon (i.v., 1 mg/kg) 5 min prior to sacrifice. Livers were homogenized at 0°C in 4vol. 0.1 M KF, 15 mM EGTA and 50 mM glycyl glycine, at pH 7.4. Following cen- trifugation at 18 800 X g for 10 min, pyruvate kinase was purified essentially as in [4] by treatment at pH 5.2, ammonium sulfate precipitation and a single chromatography on DEAE-cellulose. The enzyme was eluted from the DEAE-column (1.5 cm X 10 cm) with 100 mM potassium phosphate (pH 7.2) at a rate of 5-6 ml/min. The peak fraction was dialyzed over- night against a buffer containing 30% glycerol, 20 mM mercaptoethanol, and 100 mM Tris at pH 7.2 and stored at 5°C. When the final pyruvate kinase prepara- tion was assayed as described below at 0.15 mM PEP (1.‘6.1s), the activity was consistently