Activated endothelium releases the soluble adhesion molecules vascular cell adhesion molecule‐1 (sVCAM‐1) and intercellular adhesion molecule‐1 (sICAM‐1). Measurement of fluid‐phase adhesion molecules is therefore used to quantify endothelial activation, but it is unclear which is the better marker. The aims of the study were to compare the relationships between mRNA, surface and total expression and released VCAM‐1 and ICAM‐1 in endothelial cell cultures during activation, and to compare human umbilical vein endothelial cells (HUVEC) with the microvascular cell line HMEC‐1. sVCAM‐1 better represented mRNA and surface expression changes in HUVEC undergoing endotoxin stimulation than did sICAM‐1. Very little VCAM‐1 was released from endotoxin‐stimulated HMEC‐1, and sICAM‐1 seemed a better activation marker for these cells. During incubation of HUVEC in media with glucose concentrations of 5.6, 10.6 or 20.6 mm, VCAM‐1 was released to the media in a dose‐dependent way without changes in surface expression. ICAM‐1 was not influenced by the glucose concentration. There are situations when VCAM‐1 concentrations in the media do not mirror the surface expression on HUVEC in culture, indicating that measurements of soluble adhesion molecules may not necessarily be representative of the conditions on the cell surface. Endothelium from different locations showed varying responses with respect to VCAM‐1 and ICAM‐1 liberation upon endotoxin stimulation. Thus, both sVCAM‐1 and sICAM‐1 should be quantified in clinical studies of endothelial activation until their characteristics are better clarified.