Transendothelial migration of leukocytes is a critical event for inflammation, but the molecular regulation of this event is only beginning to be understood. PECAM (CD31) is a major mediator of monocyte and neutrophil transmigration, and CD99 was recently defined as a second mediator of the transmigration of monocytes. Expression of CD99 on the surface of circulating polymorphonuclear cells (PMN) is low compared with expression of CD99 on monocytes or expression of PECAM on PMN. We demonstrate here that, despite low expression of CD99, Fab of Abs against CD99 blocked over 80% of human neutrophils from transmigrating across HUVEC monolayers in an in vitro model of inflammation. Blocking CD99 on either the neutrophil or endothelial cell side resulted in a quantitatively equivalent block, suggesting a homophilic interaction between CD99 on the neutrophil and CD99 on the endothelial cell. Blocking CD99 and PECAM together resulted in additive effects, suggesting the two molecules work at distinct steps. Confocal microscopy confirmed that CD99-blocked neutrophils lodged in endothelial cell junctions at locations distal to PECAM-blocked neutrophils. The CD99-blocked PMN exhibited dynamic lateral movement within endothelial cell junctions, indicating that only the diapedesis step was blocked by interference with CD99. Anti-CD99 mAb also blocked PMN transmigration in a second in vitro model that incorporated shear stress. Taken together, the evidence demonstrates that PECAM and CD99 regulate distinct, sequential steps in the transendothelial migration of neutrophils during inflammation.