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We describe a modification of a previously described serum iron procedure applied to the Bayer DAX48 (Bayer Diagnostics, Tarrytown, NY) automated chemistry analyzer. The iron-ligand used in this assay, 2-(5-nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamine) phenol (nitro-PAPS), has a molar absorptivity of 94,000 L mol–1 cm–1, which is three to four times more sensitive than the more commonly used ligands. The increased sensitivity of the iron-ligand complex facilitates modification of a Ferene S method that requires a smaller sample volume while it maintains the precision of the assay. Because the reagent does not contain ascorbate, the “onboard” stability has been increased to more than 4 weeks. The reagent seems to be quite insensitive to icterus and hemolysis. Furthermore, the interference of turbidity caused by triglycerides, abnormal proteins, or fibrinogen, present in samples from patients undergoing anticoagulant therapy, seems to have been eliminated.