Addition of thallous ion (Tl+) inhibited the spontaneous mechanical activity of rat myometrium in K-free Krebs solution with an I50 value of 30 μM. The corresponding value for I50 for similar inhibition by addition of K+ was 150 μM. Tl+ as well as K+-activated p-nitrophenylphosphatase (PNPPase) of isolated rat myometrium plasma membrane vesicles. Half maximal activation was caused by 0.47 mM Tl+ or 1.6 mM K+. Maximal enzyme activities obtained using Tl+ and K+ were comparable. The Km values for the substrate p-nitrophenylphosphate using Tl+ (1.24 mM) and K+ (1.46 mM) were also similar. Activation by either ion was inhibited by ouabain, Na+, inorganic phosphate, and vanadate (V + 5). The results suggest that Tl+ can substitute for K+ for activation of the Na–K pump of rat myometrium plasma membrane.