Glycogen synthase kinase 3β (GSK-3β) negatively regulates cardiac hypertrophy. A potential target mediating the antihypertrophic effect of GSK-3β is eukaryotic translation initiation factor 2Bε (eIF2Bε). Overexpression of GSK-3β increased the cellular kinase activity toward GST-eIF2Bε in neonatal rat cardiac myocytes, whereas LiCl (10 mmol/L) or isoproterenol (ISO) (10 μmol/L), a treatment known to inhibit GSK-3β, decreased it. Immunoblot analyses using anti-S535 phosphospecific eIF2Bε antibody showed that S535 phosphorylation of endogenous eIF2Bε was decreased by LiCl or ISO, suggesting that GSK-3β is the predominant kinase regulating phosphorylation of eIF2Bε-S535 in cardiac myocytes. Decreases in eIF2Bε-S535 phosphorylation were also observed in a rat model of cardiac hypertrophy in vivo. Overexpression of wild-type eIF2Bε alone moderately increased cell size (+31±11%; P<0.05 versus control), whereas treatment of eIF2Bε-transduced myocytes with LiCl (+73±22% versus eIF2Bε only; P<0.05) or ISO (+84±33% versus eIF2Bε only; P<0.05) enhanced the effect of eIF2Bε. Overexpression of eIF2Bε-S535A, which is not phosphorylated by GSK-3β, increased cell size (+107±35%) as strongly as ISO (+95±25%), and abolished antihypertrophic effects of GSK-3β, indicating that S535 phosphorylation of eIF2Bε critically mediates antihypertrophic effects of GSK-3β. Furthermore, expression of eIF2Bε-F259L, a dominant-negative mutant, inhibited ISO-induced hypertrophy, indicating that eIF2Bε is required for β-adrenergic hypertrophy. Interestingly, expression of eIF2Bε-S535A partially increased cytoskeletal reorganization, whereas it did not increase expression of atrial natriuretic factor gene. These results suggest that GSK-3β is the predominant kinase mediating phosphorylation of eIF2Bε-S535 in cardiac myocytes, which in turn plays an important role in regulating cardiac hypertrophy primarily through protein synthesis.