Endothelium-derived relaxing factor (EDRF) released from endothelial cells (EC) has been shown to be nitric oxide (NO) or a closely related molecule. In cultured EC, the enzyme responsible for the formation of EDRF, EDRF-synthase, was initially described as being cytosolic, but more recently we have found it to be predominantly particulate. In view of this discrepancy we have investigated the EDRF synthesizing activity of cytosolic and particulate fractions isolated from native bovine aortic EC. EDRF was measured by cGMP formation in rat fetal lung cultured fibroblasts (RFL-6) and by the ability of cell fractions to relax endothelium-denuded, preconstricted rabbit aortic strips. Cytosolic fractions from native EC (100 μg) had no effect on the tone of rabbit aortic strips and little effect on cGMP levels in RFL-6 cells in the presence of L-arginine and NADPH (100 μM). However, under the same conditions the 100,000 × g pellet fractions relaxed rabbit aortic strips and increased cGMP levels in RFL-6 cells. Thus EDRF synthase from native EC, like those grown in culture, is located mainly in the particulate fraction. © 1991.