This study examined the effects of vanadate on the potassium dependent phosphatase activity present in purified human kidney microsomal (Na++K+)-adenosine triphosphatase. Vanadate anion inhibited the K+-dependent phosphatase at a KI of 35 nM. This inhibition, was noncompetitive with the substrate, p-nitrophenylphosphate. The inhibition by vanadate at 1 mM K+ was only 45% of the inhibition that was observed at 10 mM K+. Neither preincubation of the enzyme with vanadate, nor changing the pH of the assay from 8.2 to 7.2 had any effect on the KI for vanadate. The inclusion of 2.5 mM isoproterenol, to complex the yanadate, reversed the inhibition, as did diluting the enzymatic reaction. Vanadate also inhibited the overall (Na++K+)-ATPase reaction at a KI of 1.91 μM. This inhibition was also reversible upon inclusion of isoproterenol in the assay. Increasing the level of magnesium from 6 mM to 30 mM lowered the KI of vanadate to 0.25 μM. The possible role of vanadate as a physiological mediator of (Na++K+)-ATPase activity is discussed.