To study the underlying mechanism through which the endothelium-dependent relaxation is inhibited by blocking the Na⁺ -K⁺ pump, the effects of Na⁺ -K⁺ pump blockade on the release of EDRF and its relaxing activity were examined, using organ bath study, bioassay technique, and cGMP measurement. Endothelium-dependent relaxation was attenuated by blocking the Na⁺ -K⁺ pump in the vascular ring with intact endothelium. In bioassay experiment EDRF release was decreased with the blockade of the Na⁺ -K⁺ pump in the EDRF donor strip. Endothelium-dependent increase of cGMP level was suppressed by inhibiting the Na⁺ -K⁺ pump in the test strips. The magnitude of relaxation of test strip which was induced by the perfusate that had passed through the EDRF donor strip was decreased with the blockade of the Na⁺ -K⁺ pump in the test strip. Therefore, it could be suggested that the attenuation of endothelium-dependent relaxation caused by inhibiting Na⁺ -K⁺ pump activity is due to both the decreased release of EDRF from endothelial cells and the decreased sensitivity of the smooth muscle cells to EDRF.