The regulatory mechanism of prostacyclin (PGI₂) generation and its enhancement by ouabain and monensin in cultured human vascular endothelial cells was investigated with references to the kinetics of Ca⁺⁺ and Na⁺. Na⁺-K⁺ ATPase activity was assayed as ouabain-sensitive ⁸⁶Rb uptake. Cytosolic free calcium ion concentration ([Ca⁺⁺]_i) was measured using fura-2. The kinetics of ⁴⁵Ca of ²²Na, and [Ca⁺⁺]_i preceded PGI₂ generation. Ouabain inhibited Na⁺-K⁺ ATPase activity (inhibition of ²²Na release and ⁸⁶Rb uptake) and increased ²²Na uptake, while monensin enhanced ²²Na uptake and Na⁺-K⁺ ATPase activity at 2.5min after starting incubation. Both of these reagents decreased ⁴⁵Ca release and increased ⁴⁵Ca uptake (inhibition of Na⁺-Ca⁺⁺ exchange systems) at 2.5min. And all of these effects were attenuated at 10min. Ouabain and monensin increased [Ca⁺⁺]_i. Through these results it was concluded that the enhanced PGI₂ generation by ouabain or monensin was speculated to be brought about by the increased [Ca⁺⁺]_i and Ca⁺⁺ uptake which was based on the decreased Ca⁺⁺ release via the inhibition of Na⁺-Ca⁺⁺ exchange systems, and that enhanced PGI₂ generation might be autoregulated by the attenuation of increased [Na⁺]_i, which was derived from the attenuation of ouabain-induced inhibition of Na⁺-K⁺ ATPase activity, or from the enhancement of monensin-induced increase of its activity.