The interstitial fluid pressure (Pisf) has been measured in the exposed superfused mesenteries of anaesthetised rats using the micropipette servo-null technique. When mesenteries were superfused with Ringer-Locke solutions, Pisf was close to atmospheric pressure with mean ± S.E.M. values of -0.46 ± 0.14 cmH2O (n = 22). Superfusing with paraffin oil did not alter Pisf significantly, but Pisf could be lowered considerably by removing fluid from the upper surface of the mesentery. Measurements of Pisf were also made in the tissues immediately outside mesenteric venules as the pressure inside these vessels and the filtration of fluid through their walls was varied. No significant changes in perivascular Pisf could be detected even though the intravascular pressure varied from 20 to 70 cmH2O. Addition of histamine or the mast cell degranulating agent compound 48/80 to the superfusate had no significant effect on Pisf. The findings are relevant to experiments on the permeability of single perfused mesenteric microvessels. They strengthen the assumption, which is made in these studies, that Pisf is close to atmospheric pressure and does not change significantly with changes in the filtration and reabsorption of fluid through the vessel walls. Experimental Physiology (2001) 86.1, 33-38.