The effect of temperature, pH and free Mg+2 on the apparent equilibrium constant of pyruvate kinase [ATP: pyruvate-2-O-phosphotransferase] (EC 2.7.1.40) was investigated. The apparent equilibrium constant, K', for the biochemical reaction PEP + ADP = ATP + Pyr was defined as K' = [ATP][Pyr][ADP][PEP] (1) where each reactant represents the sum of all the ionic and metal complexed species in M. The K' at pH 7.0, 1.0 mM free Mg+2 and I of 0.25 M was 3.89 x 104 (n=8) at 25 °C. The standard apparent enthalpy (*H'°) for the biochemical reaction was -4.31 kJ mol-1 in the direction of ATP formation. The corresponding standard apparent entropy (*S'°) was +73.4 J K-1mol-1. The *H° and *S° values for the reference reaction, PEP3- + ADP3- + H+ = ATP4- + Pyr1- were –6.43 kJ mol-1 and +180 J K-1mol-1 respectively (5 to 38° C). We further examined the mass action ratio in rat heart and skeletal muscle at rest and found that the pyruvate kinase reaction in vivo was close to equilibrium ie. within a factor of about 3 to 6 of K' in the direction of ATP at the same pH, pMg and T. We conclude that the pyruvate kinase reaction may be reversed under some conditions in vivo, a finding which challenges the long held dogma that the reaction is displaced far from equilbrium.