Near-infrared (NIR) diffuse optical spectroscopy and imaging may enhance existing technologies for breast cancer screening, diagnosis, and treatment. NIR techniques are based on quantitative measurements of functional contrast between healthy and diseased tissue. In this study we measured the spectral dependence of tissue absorption and reduced scattering in the breasts of 30 healthy women and one woman with a fibroadenoma using a seven-wavelength frequency-domain photon migration probe. Subjects included pre- and postmenopausal women between the ages of 18 and 64. Multi-spectral measurements were used along with a four-component fit to determine the concentrations of de-oxy and oxy-hemoglobin, water and lipids in breast. The scattering spectral shape was also quantified. Our measurements demonstrate that the measured concentrations of NIR analytes correlate well with known breast physiology. Although the tissue scattering at a single wavelength was found to have little value as a functional parameter, the dependence of the scattering on wavelength provided key insights into breast composition and physiology. Lipids and scattering spectra in the breast were found to increase and decrease, respectively, with increasing body mass index. Simple calculations are also provided to demonstrate potential penalties from ignoring the contributions of water and lipids in breast measurements. Finally, water is shown to be a possible indicator for detecting a fibroadenoma, whereas the hemoglobin saturation was found to be a poor indicator. Multi-spectral measurements, compared to measurements restricted to one or two wavelengths, provide additional information that may be useful in managing breast disease. © 2002 Society of Photo-Optical Instrumentation Engineers.