Cardiac hypertrophy is characterized by increased cardiomyocyte protein synthesis, increased cell volume, and a shift in cardiac-specific gene expression to fetal isoforms. Using neonatal rat cardiomyocytes stimulated with fetal calf serum (FCS) as a model for cardiac hypertrophy, the present study investigated the role of 2 signal transduction pathways, extracellular signal-regulated kinase (ERK) and p70 S6 kinase (p70S6K), in the attendant phenotype changes. FCS evoked both ERK and p70S6K activity, peaking at 20-40 min, and simultaneously increased cardiac myocyte protein synthesis (evaluated by [³H]leucine incorporation and total cellular protein content), cell size (evaluated by morphometry and fluorescence-activated cell sorter analysis) and expression of a fetal isoform of the muscle specific gene skeletal α-actin (SKA). Rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR), which is an upstream signaling of p70S6K, completely inhibited FCS-induced cell size increases and protein synthesis, but had no effect on SKA mRNA expression. PD98059, which inhibited ERK activity, attenuated cardiac-specific gene expression in a dose-dependent manner, but had no influence on protein synthesis or cell size. These results indicate divergent roles for the ERK and p70S6K pathways in the phenotypic changes associated with cardiac hypertrophy.