Abstract—Cultured neonatal rat cardiac myocytes have been used extensively to study cellular and molecular mechanisms of cardiac hypertrophy. However, there are only a few studies in cultured mouse myocytes despite the increasing use of genetically engineered mouse models of cardiac hypertrophy. Therefore, we characterized hypertrophic responses in low-density, serum-free cultures of neonatal mouse cardiac myocytes and compared them with rat myocytes. In mouse myocyte cultures, triiodothyronine (T3), norepinephrine (NE) through a &bgr;-adrenergic receptor, and leukemia inhibitory factor induced hypertrophy by a 20% to 30% increase in [3H]phenylalanine-labeled protein content. T3 and NE also increased &agr;-myosin heavy chain (MyHC) mRNA and reduced &bgr;-MyHC. In contrast, hypertrophic stimuli in rat myocytes, including &agr;1-adrenergic agonists, endothelin-1, prostaglandin F2&agr;, interleukin 1&bgr;, and phorbol 12-myristate 13-acetate (PMA), had no effect on mouse myocyte protein content. In further contrast with the rat, none of these agents increased atrial natriuretic factor or &bgr;-MyHC mRNAs. Acute PMA signaling was intact by extracellular signal–regulated kinase (ERK1/2) and immediate-early gene (fos/jun) activation. Remarkably, mouse but not rat myocytes had hypertrophy in the absence of added growth factors, with increases in cell area, protein content, and the mRNAs for atrial natriuretic factor and &bgr;-MyHC. We conclude that mouse myocytes have a unique autonomous hypertrophy. On this background, T3, NE, and leukemia inhibitory factor activate hypertrophy with different mRNA phenotypes, but certain Gq- and protein kinase C–coupled agonists do not.