Diltiazem is a calcium antagonist widely used in the treatment of angina and hypertension. The contributions of metabolites of diltiazem to the vasorelaxant effects of diltiazem were investigated. The synthesis and spectroscopic characterization of eight major cis-diltiazem metabolites are described. Three of the compounds-N, O-didemethylated metabolite (21), O- demethylated metabolite (22), and diltiazem N-oxide (27)-have been recently reported and have not previously been synthesized. The identities of all eight synthetic metabolites have been verified with samples obtained from human urine using combined LC-MS/MS. The Ca2+ antagonistic activities of diltiazem and its metabolites (except 27) were studied on hamster arota preparations depolarized with KCl. The order of potencies (IC50 (plus or minus) SE, (mu)M) is as follows: diltiazem (0.98 (plus or minus) 0.47) > 17 (2.46 (plus or minus) 0.38) (greater-than or equal to) 23 (3.27 (plus or minus) 1.02) > 26 (20.2 (plus or minus) 10.5) > 22 (40.4 (plus or minus) 15.4) (greater-than or equal to) 25 (45.5 (plus or minus) 18.1) > 21 (112.2 (plus or minus) 33.2) (greater-than or equal to) 24 (126.7 (plus or minus) 24.2). Structure-activity relationships are also discussed.