A method is described for the partial purification of pyruvate carboxylase from rainbow trout liver. The enzyme has a pH optimum of about 8.0, possesses an absolute requirement for activation by acetylCoA, and prefers MgATP over other nucleoside triphosphates. K+ causes a decrease in the apparentK m for HCO 3 − . AcetylCoA activation shows positive cooperativity withK a=0.072 mM andn H=1.78 at pH 7.7, 2.5 mM free Mg2+, 100 mM K+, and saturating concentrations of substrates. A high acetylCoA concentration causes a decrease in the apparentK m values for MgATP and HCO 3 − and a biphasic double reciprocal plot with pyruvate as the varied substrate. MgADP and AMP are competitive inhibitors with respect to MgATP. The enzyme shows a ‘U-type’ response to the adenylate energy charge and retains considerable activity throughout a wide range of energy charge values. It is proposed that intramitochondrial acetylCoA concentration and the adenylate energy charge control the rate of pyruvate carboxylation in vivo.