Using a single reversed-phase HPLC column with a mobile phase of methanol and 100 mM phosphate buffer, concentrations of seven antibiotics (chloramphenicol, metronidazole, cefuroxime, cephalexin, ceftazidime, ampicillin and benzylpenicillin) which are commonly used clinically in combinations of two or more, can be assayed and results reported within one hour. No endogenous interference was detected in serum, urine or cerebrospinal fluid. The method was highly specific even in the presence of other drugs and metabolites commonly found in clinical samples and provides a rapid, simple technique suitable for use in routine microbiological practice. © 1990 by The British Society for Antimicrobial Chemotherapy.