The neurological disorder familial hemiplegic migraine type II (FHM2) is caused by mutations in the α2-isoform of the Na(+),K(+)-ATPase. We have studied the partial reaction steps of the Na(+),K(+)-pump cycle in nine FHM2 mutants retaining overall activity at a level still compatible with cell growth. Although it is believed that the pathophysiology of FHM2 results from reduced extracellular K(+) clearance and/or changes in Na(+) gradient-dependent transport processes in neuroglia, a reduced affinity for K(+) or Na(+) is not a general finding with the FHM2 mutants. Six of the FHM2 mutations markedly affect the maximal rate of phosphorylation from ATP leading to inhibition by intracellular K(+), thereby likely compromising pump function under physiological conditions. In mutants R593W, V628M, and M731T, the defective phosphorylation is caused by local perturbations within the Rossmann fold, possibly interfering with the bending of the P-domain during phosphoryl transfer. In mutants V138A, T345A, and R834Q, long range effects reaching from as far away as the M2 transmembrane helix perturb the function of the catalytic site. Mutant E700K exhibits a reduced rate of E(2)P dephosphorylation without effect on phosphorylation from ATP. An extremely reduced vanadate affinity of this mutant indicates that the slow dephosphorylation reflects a destabilization of the phosphoryl transition state. This seems to be caused by insertion of the lysine between two other positively charged residues of the Rossmann fold. In mutants R202Q and T263M, effects on the A-domain structure are responsible for a reduced rate of the E(1)P to E(2)P transition.