Pyruvate kinases from the kidney and liver of the osteoglossid Arapaima gigas were partially purified and characterized kinetically. The two enzymes have different elect rophoretic mobilities at pH 7.0, and while they share some qualitative similarities they show quantitative differences in their catalytic and regulatory properties. Both enzymes are activated by fructose 1.6-bisphosphate and inhibited by low levels of alanine and MgATP. The liver isozyme shows hyperbolic phosphoenolpyruvate binding, with a K1 for alanine inhibition of 0.7 mM and a K1 for MgATP inhibition of 1.0 mM. In contrast, the kidney isozyme shows cooperative phosphoenolpyruvate binding, which is accentuated at low levels of ADP. MgATP inhibition does not increase the cooperativity and shows an apparent K1 of 1.68 mM. The inhibition of alanine leads to considerable increases in the cooperativity and is effective at 1 mM and lower levels. Fructose 1.6-bisphosphate completely reverses the inhibition by alanine for both isozymes, while only leading to a partial reversal of the MgATP inhibition. These regulatory properties of both the kidney and the liver isozymes suit them for function in tissues which undergo both glycolysis and gluconeogenesis.