Elevated interstitial fluid pressure (IFP) in solid tumors may reduce the effect of systemically administered anticancer drugs. Modulation of the tumor extracellular matrix might reduce the elevated IFP. To study the influence of the microenvironment, the IFP was measured in human osteosarcoma xenografts grown both subcutaneously and orthotopically. The IFP response was recorded in xenografts grown at both sites after direct intratumoral injection of bovine testicular hyaluronidase (500 or 1600 units in 50 microliters saline). Control tumors received 50 microliters saline alone or 10% bovine serum albumin in saline. IFP was measured centrally in the tumors using the wick-in-needle technique, and mean arterial blood pressure was monitored after carotid cannulation. Tumor tissue sections were stained with hyaluronectin and analyzed for hyaluronan content using confocal laser scanning fluorescence microscopy. The baseline IFP was significantly higher in orthotopic (30 +/- 9 mmHg, n = 30) compared with subcutaneous tumors (17 +/- 6 mmHg, n = 11) of comparable sizes (p < 0.001). Injection of hyaluronidase reduced the IFP in both tumor models to 61-81% compared with controls 1 h after injection (p < 0.05), without affecting the mean arterial blood pressure significantly. The hyaluronan staining intensity increased in subcutaneous tumor sections, but remained unchanged in orthotopic tumor sections 1 h after injection of 1600 units of hyaluronidase. The IFP was restored within 48 h after hyaluronidase injection. Interestingly, IFP increased with tumor volume in orthotopic tumors, but not in subcutaneous tumors. In conclusion, intratumoral hyaluronidase injection reduces the IFP transiently in solid osteosarcoma xenografts. Furthermore, this study emphasizes that physiological parameters might differ significantly between human osteosarcoma xenografts grown subcutaneously versus orthotopically in nude mice.