Circulating soluble E-selectin is increased in diseases associated with endothelial apoptosis such as sepsis and acute respiratory distress syndrome. We investigated the mechanism by which endothelial cell (EC) apoptosis may promote soluble E-selectin release. We found that serum deprivation of EC caused apoptosis, yet it did not induce E-selectin EC surface expression. Tumor necrosis factor-alpha (TNFalpha) significantly increased EC E-selectin surface expression. Soluble E-selectin was noted, however, only in the medium of TNFalpha-activated, apoptotic EC. Preincubation of the EC with the caspase inhibitor z-VAD-fmk significantly attenuated soluble E-selectin levels in the culture medium of TNFalpha-activated, apoptotic EC, but it had no effect on E-selectin surface expression. These results indicate that TNFalpha activation, but not apoptosis, is necessary for E-selectin surface expression in EC. Furthermore, E-selectin release from EC requires caspase-3 activation. Thus, increased concentrations of circulating E-selectin in serum may serve as a marker for endothelial apoptosis in certain disease states.