Abstract： An improved approach for determination of Na+-K+-ATPase activity in single proximal renal tubule of rat reported by Doucet and his colleagues has been suggested. The single proximal renal tubules were isolated by hand under stereomicroscope from collagenase Ⅱ-treated renal cortical tissue in rats. The length of every single renal tubule segment obtained was measured. The single proximal renal tubules were treated with a hypoosmotic solution and with freeze-thaw successively, before incubation with [γ-32P]-ATP. 32 Pi hydrolyzed from [γ-32P] ATP by Na+-K+-ATPase in the single proximal renal tubules was assayed by liquid scintillation counting. The activity of Na+-K+-ATPase in the single proximal renal tubules was calculated by applying a modified formula. There was no significant difference in the measurement result of Na+-K+-ATPase activity between the method of Doucet et al. And the improved one, but the latter has advantage of less time, less reagents and being easy to operate.