Delta-like ligand 4 (Dll4) is a ligand for the membrane bound receptor Notch1. DLL4 is activated during angiogenesis when VEGF induces VEGFR2 signalling, resulting in transcellular signalling to prevent hyper sprouting. Growing blood vessels are generally leaky during pathogenic angiogenesis, and VEGF induces increased vascular permeability by increasing transendothelial transport pathways. We therefore hypothesized that the induction of the Dll4/Notch signalling pathway could provide a mechanism for this increase in permeability and that activation of the DLL4 pathway with a soluble DLL4 ligand could result in vessels with increased permeability to water (Lp: hydraulic conductivity). We used terminally anaesthetised (2% gaseous isoflurane) male Han-Wistar rats and measured hydraulic conductivity (Lp) using the modified Landis-Michel technique in post-capillary venules. Selected vessels within the rat mesentery were perfused with the soluble, extracellular portion (amino acid 26 – 524) of Dll4 recombinant protein (sDll4; 1μg/ml) in a 1% bovine serum albumin (BSA) mammalian Ringer solution containing washed red blood cells. The vessel was occluded at regular intervals and the velocity of the red blood cells, vessel radius and the length between the measured red blood cell and the occlusion point were used to calculate the transcapillary water flow per unit area of the capillary wall (Jv/S). The difference between vessel and interstitial fluid hydrostatic and osmotic pressures (ΔP) was also calculated and Lp was determined from the Starling equation: Lp = (Jv/S)/Δ P Within 120sec of perfusing with sDll4, Lp began to decrease until it plateaued at approximately 300sec decreasing from 1.8 ± 0.542 SEM to 0.41 ± 0.055 SEM x 10-7cm.s-1.cmH2O-1. Recordings taken during this nadir response period showed a 0.35 (±0.01 SEM, n=5) fold change relative to baseline. This was significantly different from perfusing with BSA alone, (p<0.001, unpaired t-test) which resulted in no change in permeability relative to baseline (1.02 fold ±0.04 SEM, n=5). We confirmed that sDLL4 activated the Notch signalling pathway in endothelial cells in culture, by measuring the expression of Hey1, a gene known to be upregulated by Notch activation. sDLL4 resulted in a 26.64 ±19.88 SEM fold increase in Hey1 expression relative to GAPDH in endothelial cells treated with 1µg/ml sDLL4 for 24 hours. Our data shows, in contrast with our hypothesis, that Dll4/Notch signalling reduces vascular permeability to water. This indicates that DLL4 activation does not contribute to the permeability induced during angiogenesis, and that potentially it could prevent increased permeability during normal, physiological angiogenesis.