Formation of a colored complex between ferric iron and xylenol orange has been used in a variety of applications, but there is disagreement about the number of complexes that exist, their stoichiometry, pH dependence, solvent effects, and other parameters. In this study we investigated the use of the complex to measure micromolar concentrations of iron in aqueous and alcohol solutions. The results show that the optimum wavelength for the measurement is 560 nm, that only a 1:1 ferric:xylenol orange complex forms, and that its molar absorption coefficient is affected by the solvent, the source of the xylenol orange, and the pH. Under the recommended conditions, formation of the complex is completed in less than 5 min and it is stable in several solvents. Its molar absorption coefficient in 25 mM H2SO4 is 20,100 or 14,500 M-1 cm-1, depending on the source of the dye. A recommended protocol for the use of the assay is given.