In veterinary medicine, lomustine has been successfull used primarily for the treatment of resistant lymphoma and also for the treatment of mast cell tumors, intracranial meningioma, epitheliotropic lymphoma, and histiocytic sarcoma in dogs either alone or in combination with other chemotherapeutic agents. Even though lomustine is commonly used in dogs primarily for the treatment of resistant lymphoma, there is no pharmacokinetics information available regarding this compound in dogs. In the present study, we developed and validated a simple high-performance liquid chromatography (HPLC) method with a one-step liquid-liquid extraction procedure to detect and quantify lomustine and its two monohydroxylated metabolites (trans- and cis-4'-hydroxylomustine) in canine plasma for future pharmacokinetic studies. The HPLC-diode-array detection method reported here readily detects lomustine, cis-4'-hydroxylomustine, and trans-4'-hydroxylomustine in canine plasma with a limit of detection of lomustine, cis-4'-hydroxylomustine, and trans-4'-hydroxylomustine in plasma of about 10 ng/120 microL, 5 ng/120 microL, and 5 ng/120 microL, respectively. The mean extraction efficiency values for lomustine, cis-4'-hydroxylomustine, and trans-4'-hydroxylomustine were 73%, 90%, and 89%, respectively, from canine plasma samples on HPLC. The present study also provides stability information about lomustine and its two monohydroxylated metabolites in canine plasma and methanol solution stored at various conditions.