A direct colorimetric method is presented for the determination of serum iron in 0.1-ml sized samples, using a new, water-soluble, reagent, 2-(5-Nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol Na salt (epsilon 585 nm = 9.4 X 10(4) l/mol per cm). Interference of copper and zinc in sera can be eliminated entirely by forming copper- and zinc-thioglycollate complexes immediately upon the dissociation of the protein-bound iron, copper and zinc by thioglycollate and sodium dodecyl sulfate. The serum blank was minimized by the use of sodium dodecyl sulfate as a protein denaturant. Within-run and between-run precision (CV) were in the range of 0.7-2.9% and 1.1-3.6%, respectively, depending on the serum iron content. A good correlation (r = 0.995) was obtained between this method and the reference method proposed by the International Committee for Standardization in Hematology.