Objective To investigate the spatiotemporal expression of connexin 43(Cx43) in the cultured ventricular myocardial cells of neonatal rats. Methods The techniques of Immunocytochemistry（ICC） and immunoelectron microscopy were used to detect the Cx43 expression in the cultured rat ventricular myocardial cells on the 2nd,4th,8th, 10th,12th,16th,20th,26th and 30th days. Results Cx43 expression was detected in the cultured ventricular myocardial cells on the 2nd day,and the Cx43 granules were located largely in the cellular cytoplasm and membrane.The punctiform granule of the cellular cytoplasm decreased and the expression of Cx43 was located mainly in cellular membrane junction on the 4th day.The expression of Cx43 increased in cellular membrane junction on the 10th day,and the morphology of Cx43 expression was chain_and strip_like. There were not obvious changes in the following days.The expression of Cx43 on the 30th day was derangement. Conclusion The spatio-temporal expression of Cx43 in the cultured ventricular myocardial cells of neonatal rats changed with the cultural time in terms of location and quantity. It was in accordance with the growth and development of the cultured