A simple isocratic HPLC procedure was developed for the analysis of seven quinolones and their metabolites in clinical specimens. It is likely that this system can be used for the assay of many other quinolone compounds, but not for nalidixic acid and the ciprofloxacin metabolite sulpho-ciprofloxacin which appear to be adsorbed in the column. Sample preparation takes approximately 20 min, and HPLC analysis is completed within 15 min with a simple solvent system eluted on a reversed phase column. The procedure is rapid, sensitive and specific, and is a modification of an assay for chloramphenicol and beta-lactam antibiotics. This method is therefore particularly useful for clinical laboratories, since a single HPLC system can be used for assays of chloramphenicol, beta-lactams and quinolones.