Nitroxoline (8-hydroxy-5-nitroquinoline) and the structural related compounds 8-hydroxyquine, 8-hydroxy-5,7-dinitroquinoline, and 8-hydroxy-5-nitrosoquinoline have been investigated by reversed-phase high-performance liquid chromatography on C18/ODS. Complete separation and symmetric, peaks were obtained by use of THF-methanol-water, 3∶3∶4, containing 10 mmol L−1 disodium ethylenediamine tetraacetic acid (EDTA) and 10 mmol L−1 citric acid in the water, as mobile phase. The pH and the concentration of EDTA in the mobile phase were found to be critical for eliminating tailing and for full separation. The calibration plot was linear for concentrations between 3.0 and 300 μg mL−1; the regression coefficient was 0.99996. Assay of the nitroxoline standard showed that recovery was from 99.3 to 102%, with a mean standard deviation for nitroxoline of 0.9%. The method is suitable for quality control of nitroxoline.