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In this study, we investigated whether calbindin-D9k (CaBP-9k) expression was regulated by melatonin during hydrogen peroxide (H2O2)-induced cell death in rat pituitary GH3 cells. CaBP-9k expression was increased by melatonin in a dose- and time-dependent manner, indicating that CaBP-9k expression is regulated by melatonin. Cell survival was increased approximately 27–30% where H2O2-treated cells (0.25 or 0.5 mm) were also incubated with 1 mm melatonin, when compared with H2O2 alone or H2O2 plus 0.5 mm melatonin. This result was consistent with 4,6-diamidino-2-phenylindole staining. CaBP-9k expression was also augmented by co-treatment with H2O2 and 1 mm melatonin, suggesting a functional relationship between increased cell death and melatonin-induced CaBP-9k expression during H2O2-mediated apoptosis. Bcl-2-associated protein expression increased following treatment with H2O2 alone, whereas Bcl-2 expression was elevated following treatment with melatonin alone, or H2O2 plus melatonin. The expression of p53 was depressed by treatment with melatonin alone, or co-treatment with H2O2 plus melatonin. These results correlated with CaBP-9k expression levels and activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway. Knockdown of CaBP-9k expression using a small inhibitory RNA resulted in an elevation of H2O2-induced cell death, whereas cell survival was increased in cells that overexpressed CaBP-9k, providing additional evidence that the induction of CaBP-9k expression may be associated with survival signaling during H2O2-mediated oxidative cell death. CaBP-9k appears to interact with p53, suggesting a possible role for this interaction in cell proliferation and cell cycle progression.