Tl-201 administration for cardial scan using SPECT has been performed for more than 4 decades, while in Iran it has been produced and used for 17 years. Although this radiopharmaceutical is injected at sub-pharmacological doses to the patients, there has been no documented research on the short-term effects on the tissues such as heart and liver, according to the best of our knowledge. In this work, the radiopharmaceutical effects on hepatic serum factors such as, billirubin (BN), alkaline phosphatase (AP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) have been investigated. The experiments were performed for periods of 1, 4 and 24 hours post injection of the tracer to 12 rats in each group in comparison with the negative control group. The results demonstrated a slight serum BN difference in two groups, while significant differences in serum AST, ALT and AP were detected. The results of this study offer some valuable information on the serum biochemical factors and Tl-201 administration relationship which is not only important in the interpretation of the clinical biochemistry tests, but also would impose limitations on the application of this tracer in patients with hepatic disorders. Further investigations on the human patients must be conducted.