Positron emission tomography studies with oxygen-15-labeled water provide in vivo quantitative tissue perfusion variables - blood flow and fractional volume of distribution of water [Vd (water)]. To investigate the relationship between perfusion variables and the effect of vascular- targeting agents on vasculature, we measured tissue perfusion in tumors, spleen, kidney, and liver before and after treatment with combretastatin-A4-phosphate, a combination of nicotinamide and carbogen (N/C), and interferon (IFN). We observed that mean tumor blood flow and Vd (water) was lower than in kidney, liver, and spleen at baseline. Blood flow and Vd (water) were related in tumor (r = 0.62; P = 0.004) at baseline, but not in other normal tissues evaluated, where minimal variations in Vd (water) were observed over a wide range of blood flow. Despite the relationship between blood flow and Vd (water) in tumors before intervention, vascular-targeting agent-induced changes in these perfusion variables were not correlated. In contrast, changes in blood flow and Vd (water) correlated in kidney and spleen after N/C and in kidney after combretastatin-A4-phosphate. The close relation between blood flow and V d (water) in tumors but not normal tissue may reflect barriers to fluid exchange in tumors because of necrosis and/or increased interstitial fluid pressure and underlies the importance and interdependence of these positron emission tomography perfusion variables under these conditions. As blood flow and Vd (water) signify different aspects of tissue perfusion, the differential effects of interventions on both variables, flow and Vd (water), should therefore be reported in future studies. Copyright © 2009 American Association for Cancer Research.