The antibacterial activity of nitroxoline (NIT), an antibiotic used in the treatment of acute or recurrent urinary tract infections caused by Escherichia coli, is decreased in the presence of Mg2+ and Mn2+ but not Ca2+. In order to elucidate the interaction between this drug and the divalent cations, spectrophotometric studies based on the natural absorption of the nitroxoline moiety were conducted. In the presence of the divalent metal ions, a shift in the NIT A448 suggested the formation of drug-ion complexes, for which the stability followed the order Mn2+ > Mg2+ > Ca2+. A clear correlation was found between the chelating property and antibacterial activity of NIT; both were pH dependent. A convenient colorimetric method for the determination of NIT uptake by bacterial cells was also developed. Uptake was energy independent and showed biphasic kinetics: a rapid association with cells and then a slower increase in cell-associated NIT which reached a plateau. NIT uptake was reduced in the presence of magnesium. The implications of metal ion complexation and pH on the clinical efficacy of NIT are discussed.