The analytical conditions used in the determination of diltiazem (D) and deacetyldiltiazem (DAD) in post mortem blood using high-performance liquid chromatography (HPLC) are presented and a simple method of preparingDADis described. These xenobiotics were isolated from blood by liquid-liquid extraction (LLE) in a moderately alkaline medium with application of a mixture of dichloromethane and ether and determined with a liquid chromatograph (Gilson), using a Hypersil ODS (250 x 4 mm, 5 μm) column, a mobile phase of 1.5 ml/min flow (in an isocratic system of two pumps) consisting of acetonitrile and phosphate buffer at pH = 3 (0.025 M solution of phosphoric acid with addition of 6 ml of triethylamine per 1 litre) in proportions 25:75 and UV detection at 235 nm. Good separation of D and DAD from post mortem background was achieved and the average recovery from blood was 78% and 71% respectively. The level of detection of both xenobiotics was about 0.5 μg/ml (with a coefficient of variation CV within the range 2.6-9.1%). The method did allow us to determine concentrations that were twice as low (0.25 μg/ml), but at the cost of a decrease in precision (CV = 16.0-17.8%).