Five aminoglycoside antibiotics (AGAs) – kanamycin (KM), bekanamycin (AKM), dibekacin (DKB), ribostamycin (RSM) and paromomycin (PRM) – were studied for their effects on the nonenzymic reduction of cytochrome c by FeSO4 PRM. As this order correlated closely with that of the antibacterial activity of AGAs, Yamabe’s system has proved useful in predicting the latter activity. Divalent metal ions other than Fe2+ enhanced the AGA-dependent inhibition of Yamabe’s system in the order: Cu2+ Mn2+ Zn2+ Co2+ Ni2+ . This order was similar to that of stability constants with general chelators except for the low positions of Ni2+ and Co2+. These findings suggested a metal chelation with free or bound Fe2+ for the action mechanism of AGAs on Yamabe’s system and the bacterial growing system. The antagonistic effects of exogenous Fe2+ on the antibacterial activity against Staphylococcus aureus and Escherichia coli as measured by the agar dilution method supported this concept. A dual relationship of molecular structure with chelating and antibacterial activities demonstrated the importance of high molecular basicity in a potent AGA. However, the combination effect of pipemidic acid (stimulator on Yamabe’s system) with KM was different from that with 1,10-phenanthroline (inhibitor on Yamabe’s system) as measured by Dye’s method using Pseudomon