Haemoglobin oxygen saturation in subcutaneous rat mammary tumours was measured using near infrared spectroscopy (NIRS) in vivo and in rapidly frozen sections from the same tumours using cryospectrophotometry, which reports oxygen saturation in individual blood vessels to depths of 4 mm from the tissue surface. Measurements were performed on tumours while animals breathed either room air or carbogen. In five of nine tumours, the average saturation calculated from cryospectrophotometric measurements agreed with that determined from NIRS to within 13%, and in four of these five tumours agreement was 5% or better. In the remaining four of nine tumours, where agreement was poor, the volume-averaged saturations estimated from NIRS were consistently higher than those calculated from cryospectrophotometry. Monte Carlo simulations demonstrated that the depth of tissue probed by NIRS was significantly greater than that sampled by cryospectrophotometry. Analysis of the frequency of severely hypoxic vessels showed that when NIRS reported a saturation of approximately 70% or higher, the fraction of tumour vessels with saturations less than 10% was limited to 0.06 or less. Sensitivity and specificity analysis suggests that NIRS and NIRS imaging may identify clinically relevant hypoxia, even when its spatial extent is below the resolution limit of the NIRS technique.