The shift in optical absorption of hemoglobin upon binding of oxygen provides a basis for near-infrared monitoring of hemoglobin oxygen saturation, which is an important indicator of tissue oxygenation. Tumor oxygenation has long been studied, because hypoxic cells exhibit resistance to ionizing radiation therapy. The ability to measure noninvasively the oxygenation status of tumors and their response to oxygen modifiers is important in research and clinical settings. We have implemented a steady-state diffuse reflectance method of optical spectroscopy in scattering systems based on the theory of Farrell et al. (Med. Phys., 1992). In scattering phantoms containing erythrocytes, the method recovers the hemoglobin absorption spectrum (650 - 820 nm) and accurately monitors hemoglobin oxygen saturation. We have implemented a probe that individually positions several detection fibers normal to the surface of subcutaneous rodent tumors. Near-infrared absorption spectra reconstructed from diffuse reflectance measurements indicate a hemoglobin oxygen saturation of approximately 50% in R3230AC rat mammary adenocarcinomas when the anesthetized animal breathes room air. Administration of carbogen (95% oxygen, 5% carbon dioxide) via a nose cone produces a rapid and readily detectable increase in the saturation to 75% with no increase in tumor blood volume. Several methods of determining hemoglobin oxygen saturation from absorption spectra obtained by diffuse reflectance spectroscopy are compared, including singular value decomposition, which provides the ability to reconstruct the non-hemoglobin absorbing background without a priori knowledge of its structure or absolute magnitude.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.