Endothelial damage during early endotoxemia has been shown to be leukocyte independent. Platelet-activating factor and serotonin receptor antagonism has been shown to reduce leukocyte-independent macromolecular leakage significantly. Nevertheless, the exact mechanisms involved in leukocyte-independent endothelial dysfunction are unknown. Therefore, it was the aim of the study to investigate the effects of nitric oxide (NO) on leukocyte-independent endothelial damage during endotoxemia. In male Wistar rats, venular wall shear rate, macromolecular efflux, and leukocyte-endothelial interaction were determined in mesenteric postcapillary venules using intravital microscopy at baseline and at 60 and 120 min after start of the experiment. The animals received fucoidin to prevent leukocyte-endothelial interaction. The experiments were divided into three parts. In part 1, we investigated the effects of the NO-inhibitor L-NAME on leukocyte-independent endothelial damage during endotoxemic and nonendotoxemic conditions. The efficiency of the NO-donor (SIN-1) used, part 2, was investigated by the inhibitory properties of SIN-1 on NO-inhibition-induced macromolecular efflux. Finally, part 3, we analyzed the effects of the NO-donor SIN-1 on endothelial damage during endotoxemia. Both the combined challenge of the animals with L-NAME and endotoxin and the challenge with L-NAME alone resulted in a strong increase in macromolecular efflux, showing significant differences to control groups at an earlier time point than endotoxin challenge alone. Interestingly, combined L-NAME and endotoxin challenge, L-NAME challenge alone, and endotoxin challenge alone showed a similar macromolecular efflux at the end of the experiment. SIN-1 prevented both the increase in macromolecular efflux seen after L-NAME challenge (part 2) and was highly effective in preventing significantly the increase in macromolecular leakage that is seen during leukocyte-independent endotoxemia (part 3). In conclusion, our data indicate that during early states of endotoxemia endogenous NO preserves endothelial integrity in a leukocyte-independent setting. Exogenous NO prevents endothelial damage during early leukocyte-independent endotoxemia. Summarizing these data, endothelial integrity during leukocyte-independent endotoxemia is a NO-mediated event.