An isocratic high-performance liquid chromatographic method with detection at 240 nm was developed, optimized and validated for the determination of ketoconazole in canine plasma. 9-Acetylanthracene was used as internal standard. A Hypersil BDS RP-C18 column (250 mm x 4.6 mm, 5 microm particle size), was equilibrated with a mobile phase composed of methanol, water and diethylamine 74:26:0.1 (v/v/v). Its flow rate was 1 ml/min. The elution time for ketoconazole and 9-acetylanthracene was approximately 9 and 8 min, respectively. Calibration curves of ketoconazole in plasma were linear in the concentration range of 0.015-10 microg/ml. Limits of detection and quantification in plasma were 5 and 15 ng/ml, respectively. Recovery was greater than 95%. Intra- and inter-day relative standard deviation for ketoconazole in plasma was less than 3.1 and 4.7%, respectively. This method was applied to the determination of ketoconazole plasma levels after administration of a commercially available tablet to dogs.