Aim: To investigate whether hypoxia reoxygenation induces premature senes-cence in neonatal Sprague-Dawley (SD) rat cardiomyocytes. Methods: Cardio-myocytes were isolated from neonatal SD rat heart and identified by immunohisto-chemistry. The control cultures were incubated at 37 ℃ in a humidified atmo-sphere of 5% CO<,2> and 95% air. The hypoxic cultures were incubated in a modular incubator chamber filled with 1% O2, 5% CO2, and balance N2 for 6 h. The reoxygen-ated cultures were subjected to 1% O2 and 5% CO2 for 6 h, then 21% oxygen for 4,8, 12, 24, and 48 h, respectively. Cell proliferation was determined using bromo-deoxyuridine labeling. The ultrastructure of cardiomyocytes was observed by using an electron microscope. Β-Galactosidase activity was determined by using a senescence β-galactosidase Staining Kit. P16INK4a and telomerase reverse tran-scriptase (TERT) mRNA levels were measured by real time quantitative PCR. TERT protein expression was determined by immunohistochemistry. Telomerase activi-ties were assayed by using the Telo TAGGG Telomerase PCR ELISApplus kit. Results:The initial cultures consisted of pure cardiomyocytes identified by immunohisto-chemistry. The proportion of BrdU positive cells was reduced significantly in the hypoxia reoxygenation-treated group (P<0.01). Under the condition of hypoxia reoxygenation, mitochondrial dehydration appeared; p16’INK4a and TERT mRNA levels, β-galactosidase activity, TERT protein expression and telomerase activi-ties were all significantly increased (P<0.01 or P<0.05). Conclusion: These data indicate that premature senescence could be induced in neonatal SD rat cardiomyo-cytes exposed to hypoxia reoxygenation. Although TERT significantly increased,it could not block senescence.