Bathophenanthrolinedisulfonic acid (4,7-diphenyl1,10-phenanthrolinedisulfonic acid [BPS]) and (3-(2-) pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine), chelators of ferrous iron, are often used to determine iron(II) concentrations in various samples and for identifying or measuring iron reduction in biological systems. In this study, the efficacy of ferrozine and BPS to chelate iron(II) reduced from (\mathrm{Fe}^{3+})-ligands by selected reducing agents was determined. Our results indicate that (i) BPS and ferrozine are not equivalent as kinetic indicators of iron reducing activity; (ii) apparent initial rates of reduction of (\mathrm{Fe}^{3+})-ligands by dithiothreitol, as indicated by formation of complexes of iron(II) with either BPS or ferrozine, differed by a factor of 50 ; and (iii) nonspecific reduction of some (\mathrm{Fe}^{3+})-ligands by both BPS and ferrozine occurred. Under identical conditions, rates of formation of (\mathrm{Fe}^{2+}-) ferrozine generally were slower than rates of formation of (\mathrm{Fe}^{2+}-\mathrm{BPS}). These data suggest careful consideration should be given in the design of any experiments where kinetics of iron reduction are monitored with BPS or ferrozine. (\subset 1993) Academic Press, Inc.