The use of the calcium antagonist diltiazem (I) is increasing, particularly in treating unstable angina. This study describes a simple and specific high-performance liquid chromatographic method for the determination of I and six of its metabolites in urine. Diltiazem and its conjugated and unconjugated metabolites were assayed in the urine of patients treated with a dose of 120 mg po of diltiazem tid. Sensitivity of the method was 100 ng/mL for I and the metabolites desacetyldiltiazem (II), N-desmethyldesacetyldiltiazem (IV), O-desmethyldesacetyldiltiazem (V), O-desmethyl-N-desmethyldesacetyldiltiazem (VII), N-oxide desacetyldiltiazem (III), and O-desmethyl-N-oxide desacetyldiltiazem (VI). In the urine of patients, the extent of conjugation of six metabolites was greater than 80%.