(Na' + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(II1) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na' + K')-ATPase activity was accompanied by a parallel decrease of the associated K +-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na '-dependently, a phosphointermediate from [y-32P]ATP.The kinetics of inactivation and of phosphorylation with [ I > -~~P ] C~A T P and [ u -~~P I C~A T P are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme : ADP E + CrATP e E . C r A T P e E c r f E c r t E + C?' + P,. The dissociation constant of the CrATP complex of the pig kidney enzytne at 37 ' C was 43 pM. The inactivation rate constant (k+Z = 0.033 min-') was in the range of the dissociation rate constant k dof ADP from the enzyme of 0.011 min-'. The phosphoenzyme was unreactive towards ADP as well as to K + . N o hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na' reactivated it slowly. 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na f-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (drssociation constant ofthe enzyme ATP complex = 2.5 pM) as well as low concentrations of K'. CrATP was a competitive inhibitor of (Na' + K+)-ATPase.It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na' + K + ) -ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent protein kinase reaction as the phosphate acceptor site. magnetic resonance studies indicate the presence of one divalent metal ion bound at the active site [lo], where the divalent cation seems to maintain the phosphointermediate in the reactive state [ll]. There is evidence that the phosphointermediate formed in the presence of Na+ is converted from an ADPsensitive state into a K t -sensitive state [l, 3,11,12]. The mechanism for this interconversion is unclear, however; the earlier assumption that free Mg2 + might be necessary [1,3,12] has been doubted [8,13,14] since the proposed cyclical changes in the affinity of Mg2' for binding to and release from the enzyme need affinity changes of at least four orders of magni-