Malignant tumors generate new blood vessels by secreting growth factors, particularly members of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) family. Overall, the new blood vessels that form are hyperpermeable to plasma proteins, a property that is thought to be important for generating new stroma. However, tumor blood vessels are structurally heterogeneous and include microvessels of at least the following distinct types: mother vessels (MV), glomeruloid microvascular proliferations (GMP), arterio-venous-like vascular malformations and capillaries. Our goal was to determine whether macromolecular tracers leaked from all or from only a subset of these vessel types and to elucidate the extravasation pathways. As blood vessels are only a minor component of tumors, and therefore, difficult to study in situ, we used an adenoviral vector to express VEGF-A164, the most important member of the VPF/VEGF family, in mouse tissues. So expressed, VEGF-A164 induces large numbers of surrogate vessels of each type found in tumors in a highly reproducible manner. Overall permeability to plasma proteins was assessed qualitatively with Evan's blue dye and quantitatively with a dual tracer method employing radioactive albumin. Leaky vessels were identified by confocal microscopy (FITC-dextran) and by electron microscopy (ferritin). MV, and to a lesser extent GMP, were found to be hyperpermeable but capillaries and vascular malformations were not. Ferritin extravasated primarily by two trans-cellular routes, vesiculo-vacuolar organelles (VVOs) and fenestrae. This occurred despite a considerable reduction in VVO frequency as VVO membranes translocated to the plasma membrane during MV formation. However, reduction in the number and complexity of VVOs was offset by extensive endothelial cell thinning and a greatly shortened extravasation pathway. Extrapolating these findings to tumors predicts that only a subset of tumor vessels, MV and GMP, is hyperpermeable, and that measures of overall vessel permeability greatly underestimate the permeability of individual MV and GMP.