Red blood cells are widely accepted to be inert carriers of oxygen and haemoglobin, but there is growing evidence that they play a much more critical role in immune function. Macrophage migration inhibitory factor (MIF) is a key cytokine in disease with additional oxido-reductase activity, which aids in managing oxidative stress. Although two studies have reported the presence of MIF in red blood cells, no study has quantified the levels of this protein. In this study, freshly isolated plasma, platelets, leukocytes, and red blood cells from healthy individuals were collected and the concentration of MIF was determined using an enzyme linked immunosorbent assay. This analysis demonstrated that MIF in red blood cells was present at 25 μg per millilitre of whole blood, which is greater than 99% of the total MIF and 1000-fold higher concentration than plasma. This result was supported by electrophoresis and Western blot analysis, which identified MIF in its monomer structural form following sample processing. Furthermore, by assessing the level of tautomerase activity in red blood cell fractions in the presence of a MIF inhibitor, it was determined that the red blood cell-derived MIF was also functionally active. Together, these findings have implications on the effect of haemolysis during sample preparation and provide some clue into the inflammatory processes that occur following haemolysis in vivo. These results support the hypothesis that red blood cells are a major reservoir of this inflammatory protein and may play a role in inflammation.