A dog histopathologically diagnosed with hepatocellular carcinoma (HCC) showed very high serum alkaline phosphatase (ALP) activity. A supernatant of ascitic fluid and tumor tissue extracted from the dog also showed much higher ALP activity than normal. ALP isoenzyme analysis of samples was performed using polyacrylamide gel disk electrophoresis, and a wide, broad abnormal band was observed. By various treatments, the abnormal band showed thermostability, which is a characteristic of tumor-associated ALP that has only been reported in humans. The thermostable ALP isoenzyme was not found in sera from 39 dogs with several types of tumor that originated from the liver, except for HCC, nor was it found in 10 dogs with hepatic diseases that did not include hepatic tumors. The thermostable ALP isoenzyme seemed to be associated with canine HCC.