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Background: In addition to the classical adrenal cortical biosynthetic pathway, there is increasing evidence that aldosterone is produced in extra-adrenal tissues. Although we have previously reported that aldosterone is produced in the heart, the concept of cardiac aldosterone synthesis remains controversial. This is partly due to the lack of established experimental models representing the aldosterone synthase gene (CYP11B2) expression in a robustly reproducible fashion. Therefore, in this study, we investigated suitable conditions in a neonatal rat cardiomyocytes culture system producing CYP11B2 with considerable efficacy. Methods and Results: Neonatal rat cardiomyocytes were cultured in serum-free medium containing various doses of glucose for two to 24 hours. The CYP11B2 mRNA expression in the cardiomyocytes was determined using real-time PCR, and the aldosterone concentrations secreted from the cardiomyocytes into the culture medium were measured using an enzyme immunoassay. We found that suitable conditions for inducing the cardiac aldosterone gene expression included four hours of incubation with high glucose conditions (>25 mM). Under these particular conditions, the CYP11B2 expression (2.18±0.37-fold, p Conclusions: The suitable conditions for detecting the CYP11B2 expression and aldosterone secretion in the neonatal rat cardiomyocytes culture system are now clarified: high glucose conditions with a relatively brief period of culture promote the CYP11B2 expression as well as aldosterone production/secretion in cardiomyocytes. The current system will help to accelerate further progress in research on cardiac tissue aldosterone synthesis.