The effects of a new contrast agent, ioxilan, on vascular endothelium were compared with those of iohexol and diatrizoate. Rabbit aortic rings were incubated in contrast medium (CM) (350 mgI/mL) or Krebs solution as a control agent, for 5 minutes. Scanning and transmission electron micrographs showed that iohexol and ioxilan produced irregularities in the cell borders and in some vesicles, whereas diatrizoate produced intercellular gaps and numerous vesicles containing myelin figures. The ability of the endothelial cells to release endothelium-derived relaxing factor was tested by measuring the dilator response to acetylcholine. Incubation of aortic rings in CM for 5 minutes caused no changes in responses. However, 15-minute contact with diatrizoate irreversibly reduced the dilator response to 49%, and contact with sucrose (2100 mOsm/kg) reduced it to 9%. After incubation for 60 minutes, iohexol reduced the dilator response to 43%, while ioxilan caused no change. Since the hydrophilicity of the nonionic compounds, ioxilan and iohexol, is similar, while ioxilan's osmolality is substantially lower, the endothelial changes detected by electron microscopy and induced by the CM are attributable to their chemical properties, whereas the loss of dilator response appears to be mediated by high osmolality.