Endothelial cells in vivo are well known to respond to parallel shear stress induced by luminal blood flow. In addition, fluid filtration across endothelium (transendothelial flow) may trigger nitric oxide (NO)-production, presumably via shear stress within intercellular clefts. Since NO regulates neutrophil-endothelial interactions, we determined whether transendothelial flow regulates neutrophil transmigration. Interleukin-1 (IL-1)-treated human umbilical vein endothelial cell (HUVEC) monolayers cultured on a polycarbonate filter were placed in a custom chamber with or without a modest hydrostatic pressure gradient (P, 10 cmH₂O) to induce transendothelial flow. In other experiments, cells were studied in a parallel plate flow chamber at various transendothelial flows (P = 0, 5, and 10 cmH₂O) and luminal flows (shear stress of 0, 1, and 2 dyn/cm²). In the absence of luminal flow, transendothelial flow reduced transmigration of freshly isolated human neutrophils from 40% to 5% (p<0.05), and induced an increase in NO detected with a fluorescent assay (DAF-2DA). The NO synthase inhibitor, L-NAME, prevented the effects of transendothelial flow on neutrophil transmigration while a NO donor (DETA/NO, 1 mM) inhibited neutrophil transmigration. Finally, in the presence of luminal flow (1 and 2 dyn/cm²), transendothelial flow also inhibited transmigration. Based on HUVEC morphometry and measured transendothelial volume flow (Jv), we estimated cleft shear stress to range from 49-198 dyn/cm². These shear stress estimates, while substantial, are of similar magnitude to those reported by others with similar analyses. These data are consistent with the hypothesis that endothelial cleft shear stress inhibits neutrophil transmigration via a NO-dependent mechanism.