Hypoxic fractions were measured in multiple tumor models and compared to 31P magnetic resonance spectroscopy (MRS) parameters. Hypoxic fractions were measured by excision assays in intramuscular and subcutaneous RIF-1 tumors, large and small SCCVII tumors, and KHT tumors. The hypoxic fractions ranged from 3% (small SCCVII) to 21% (KHT). Measurements of 31P MRS parameters (indices of tumor metabolism) were made under the same conditions used in the hypoxic fraction assays. The PCR/Pi ratios in the five tumor models were strongly correlated (r2 = 0.98, tau = 1.00, P < 0.02) with their hypoxic fractions, and the NTP/Pi ratio was weakly correlated (r2 = 0.67, tau = 0.60, P < 0.17) with hypoxic fraction. Smaller values of the PCr/Pi and NTP/Pi ratios were found in the tumors with larger hypoxic fractions. These experiments indicate that 31P MRS may provide a relative measurement of hypoxic fractions across different tumor lines that can be applied non-invasively to individual tumors.