The barrier function of blood vessels is though to be regulated at least in part by endothelium. This concept is supported by the dramatic loss of barrier function occurring in the hyperacute rejection of vascularized grafts mediated by anti-endothelial cell (EC) antibodies and complement. In this process, the endothelium is not destroyed but instead loses the ability to retain blood cells and plasma proteins within capillaries. The noncytotoxic mechanism that allows this change in EC function has been unknown. Here we report that within 10 to 20 min of exposure to human xenoreactive natural antibodies and complement, porcine EC undergo alterations in cell shape and in the cytoskeleton that disrupt monolayer integrity and lead to formation of intercellular gaps. Gap formation is not associated with cell death but requires the complement complex C5b67. The gaps induced by anti-EC antibodies and complement are transient; gap closure requires formation of C5b-9 complexes on the cells and the rate of recovery depends on the release of cellular products into the medium. Preincubation of EC with dibutyryl cAMP (0.5 mM) prevents gap formation and disruption of the cytoskeleton caused by antibodies and complement. These results provide evidence that the integrity of endothelium is regulated by components of the complement system and suggest a mechanism that may explain the prominent loss of endothelial integrity seen in humoral immune responses.