This paper describes the determination of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil in urine. The method involves sample clean-up by liquid-liquid extraction with ethyl acetate followed by high-performance liquid chromatographic (HPLC) analysis. The sample preparation may be performed either manually or automatically using a Zymark Py-robotic system. The chloro analog of the parent compound, CV-araU, is used as the internal standard. As low as 0.1 microgram of analyte per ml of urine can be measured. This sensitivity is adequate for pharmacokinetic studies but could be improved quite easily if necessary.