A bioassay technique and organ bath study were performed to analyze the effects of extracellular Ca2+ and Ca2+-antagonists on endothelium-derived relaxing factor[s][EDRF] released from the endothelial cells of rabbit aorta. Transverse strips with intact endothelium or damaged endothelium were used for the mechanical contraction experiment using organ bath. Long segment including thoracic and abdominal aorta with endothelium [EDRF donor aorta] was perfused with Tyrode solution which was aerated with 95% O2 + 5% CO2 mixed gas and kept at 35oC. The perfusate was bioassayed with a transverse strip of thoracic aorta with damaged endothelium. The test strip was contracted with nor-epinephrine and acetylcholine was used to stimulate the release of EDRF from endothelial cells. The results obtained were as follows; 1] The endothelium-dependent relaxation[EDR] induced by acetylcholine was biphasic; an initial rapid relaxation followed by a slow relaxation. 2] EDR induced by acetylcholine was reduced gradually with the decrease in the concentration of extracellular Ca2+. The effect of extracellular Ca2+ on EDR was more prominent in the late slow relaxation phase. 3] EDR to acetylcholine was not altered by acute exposure to organic Ca2+-antagonists. Pretreatment with verapamil to the EDRF donor aortic segment did not alter the magnitude of EDR. 4] Among the inorganic Ca2+-antagonists Mn2+ and Cd2+ did not inhibit EDR, whereas Co2+ and La3+ inhibited EDR. 5] The inhibitory response of Co2+ to EDR developed when infused directly on the test strip. That of La3+, however, was evoked when added to solution perfusing the donor aortic segment. The above results suggest that Ca2+-antagonists do not affect EDR and the inhibitory effect of Ca2+ results from influencing the action of EDRF on vascular smooth muscle, whereas that of La3+ results from its action on the release of EDRF from endothelial cells