Purpose: Na+-K+ ATPase Activity has beem estimated by the degree of inhibition of cation transport by cardiac glycosides (ouabain) using Rb-86 as a substrate. The biological characterist- Isc of T1-201 is known to be simiIar to those of potassium as a transport substrate in the presence of glucose, insulin or phobol myristate acetate (PMA). The purpose of this study was to measure ouabain sensitive Na+-K+ ATPase activity using T1-201 and compare with that using Rb-86. Materials and Methods: Smooth muscle cells isolated from rat aorta or human placental umbilical artery were cultured, and used to measure cellular Na+-K+ ATPase activity. Na+-K+ ATPase activity was measured as a percentage decrease in cellular uptake of T1-201 or Rb-86 by ouabain under the presence of glucose, insulin or PMA in media. Results: Na+-K+ ATPase ase activity measured with T1- 201, as a transport substrate, was not different from those measured with Rb-86 in rat or human smooth muscle cell preparation. Incubation with high concentration glucose resulted in about 30% decrease in enzyme activity. In contrast, insulin or PMA resulted in 50- 70% or 28% increases from baseline activity, respectively. Conclusion: These results suggests that T1-201 could replace Rb-86 in measurement of ouabain sensititive Na+-K+ ATPase activity in vitro. High level of glucose concentration decreased cellular Na+-K+ ATPase activity, but insulin or PMA increased it.