Guanabenz (2, 6-dichlorobenzylidene-amino-guanidine) is a centrally acting antihypertensive drug whose mechanism of action is via alpha2 adrenoceptors or, more likely, imidazoline receptors. Guanabenz is marketed as an antihypertensive agent in human medicine (Wytensin tablets, Wyeth Pharmaceuticals). Guanabenz has reportedly been administered to racing horses and is classified by the Association of Racing Commissioners International as a class 3 foreign substance. As such, its identification in a postrace sample may result in significant sanctions against the trainer of the horse. The present study examined liquid chromatographic/tandem quadrupole mass spectrometric (LC-MS/MS) detection of guanabenz in serum samples from horses treated with guanabenz by rapid iv injection at 0.04 and 0.2 mg/kg. Using a method adapted from previous work with clenbuterol, the parent compound was detected in serum with an apparent limit of detection of approximately 0.03 ng/ml and the limit of quantitation was 0.2 ng/ml. Serum concentrations of guanabenz peaked at approximately 100 ng/ml after the 0.2 mg/kg dose, and the parent compound was detected for up to 8 hours after the 0.04 mg/kg dose. Urine samples tested after administration of guanabenz at these dosages yielded evidence of at least one glucuronide metabolite, with the glucuronide ring apparently linked to a ring hydroxyl group or a guanidinium hydroxylamine. The LC-MS/MS results presented here form the basis of a confirmatory test for guanabenz in racing horses.