Mesencephalic dopaminergic neurons are heavily involved in the development of drug dependence. Thyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, plays an important role in the survival of dopaminergic neurons. Therefore, this study investigated TH changes in dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN), as well as the morphine effects on dopaminergic neurons induced by different durations of morphine dependence. Models of morphine dependence were established in rats, and paraffin-embedded sections, immunohistochemistry and western blotting were used to observe the changes in the expression of TH protein. Fluoro-Jade B staining was used to detect degeneration and necrosis, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) detected the apoptosis of mesencephalic dopaminergic nerve cells. Immunohistochemistry and western blotting showed that the number of TH positive cells and the protein levels in the VTA and SN were significantly decreased in the rats with a long period of morphine dependency. With prolonged morphine exposure, the dopaminergic nerve cells in the VTA and SN showed degeneration and necrosis, while apoptotic cells were not observed. The number of VTA and SN dopaminergic nerve cells decreased with increasing periods of morphine dependence, which was most likely attributable to the degeneration and necrosis of nerve cells induced by morphine toxicity.