A microspectrophotometric imaging method has been developed for localized measurements of intravascular oxyhemoglobin (HbO₂) saturations in microvessels from sections of quick-frozen tissue. HbO₂ saturation was calculated from the absorption spectrum of red blood cells measured at five selected wavelengths in the 520- to 570-nm range. We combined the use of narrow-bandwidth interference filters and a CCD camera mounted on a microscope to obtain one gray image of the sample at each wavelength. Each pixel is a quantitative measure of transmitted light intensity from the tissue sample at that location. A linear calibration curve for blood frozen in vitro (humans and mice) and in vivo (mice) was obtained using a multicomponent analysis. Oxy- and deoxyhemoglobin were assumed to be the only hemoglobin components present. A constant term compensates for light loss due to scattering on red blood cells and ice crystals. The standard error in single measurements of HbO₂ saturation was 5%. The present method allows off-line analysis of the HbO₂ saturation distribution within a microvessel network and offers new possibilities for comparative morphological studies.