Cancer therapy based on tumor-selective macromolecules may fail due to the elevated interstitial fluid pressure (IFP) that reduces the transvascular and interstitial convection in solid tumors. Modulation of the tumor extracellular matrix (ECM) may reduce IFP and enhance transvascular filtration and interstitial transport of macromolecules. We therefore measured the effect of the ECM-degrading enzyme collagenase on IFP and microvascular pressure (MVP) in human osteosarcoma xenografts using the wick-in-needle and micropipette methods, respectively. The tumor uptake and distribution of a systemically administered osteosarcoma-associated monoclonal antibody (TP-3) after i.v. injection of collagenase were analyzed using confocal laser scanning microscopy. Collagenase (0.1%) reduced both IFP (45%) and MVP (60%), but the kinetics of the recoveries differed, because MVP had recovered by the time IFP reached its minimum level. Thus, collagenase increased the transcapillary pressure gradient, inducing a 2-fold increase in the tumor uptake and improving the distribution of the monoclonal antibody, which was localized further into the tumor. To study the mechanism of the reduction in MVP, mean arterial blood pressure was measured and found not to be affected by the collagenase treatment. The reduction in MVP was rather due to reduced vascular resistance because microvascular-associated collagen was totally or partially disintegrated. Although collagenase may favor metastasis and thus not be clinically relevant, this study shows proof of principle that degradation of the ECM leads to a favorable change in the transvascular pressure gradient, thereby increasing antibody penetration and binding to tumor cells.