Diltiazem (DTZ) is a calcium antagonist widely used in the treatment of angina and related heart diseases. It is extensively metabolized into a host of metabolites, some of which have potent pharmacological activities. In this study, the pharmacokinetics and metabolism of DTZ was investigated in humans, dogs, rabbits, and rats after each species (n = 4 or 5) was given a single oral dose of DTZ. After the drug administration, blood and urine samples were collected for 12 and 48 hrs, respectively. DTZ and six of its metabolites were quantitated in our laboratory by HPLC. The results indicated that, in humans, the major metabolites in plasma were N-monodesmethyl diltiazem (MA), deacetyl diltiazem (M1), and deacetyl N-monodesmethyl diltiazem (M2). These metabolites were also detected in the plasma of dogs, rabbits, and rats. However, there were quantitative differences. For example, in the humans and dogs, MA was the most abundant metabolite in plasma, while M1 and M2 were most prominent in the rabbits and rats, respectively, and M2 was a relatively minor metabolite in dog plasma. Less than 5% of the dose was recovered as unchanged DTZ in the urine of all the tested species. The most abundant metabolites in urine appeared to be MA and deacetyl N,O-didesmethyl diltiazem, although there were considerable inter- and intra-species variations. Two additional metabolites were detected in the urine of the humans, dogs, and rabbits, but not in the rats. They were tentatively identified as O-desmethyl diltiazem and N-O-didesmethyl diltiazem, using electron impact and ammonia chemical ionization mass spectrometry. Thus, it was concluded that the species differences of DTZ metabolism and kinetics were mainly quantitative rather than qualitative.