Signaling via mitogen-activated protein kinases is implicated in heart failure induced by agonists for G protein-coupled receptors that act via the G protein Galphaq. However, this assertion relies heavily on pharmacological inhibitors and dominant-interfering proteins and not on gene deletion. Here, we show that endogenous cardiac MAPK/ERK kinase kinase-1 (MEKK1)/(MAP3K1), a mitogen-activated protein kinase kinase kinase, is activated by heart-restricted overexpression of Galphaq in mice. In cardiac myocytes derived from embryonic stem cells in culture, homozygous disruption of MEKK1 selectively impaired c-Jun N-terminal kinase activity in the absence or presence of phenlyephrine, a Galphaq-dependent agonist. Other terminal mitogen-activated protein kinases were unaffected. In mice, the absence of MEKK1 abolished the increase in cardiac mass, myocyte size, hypertrophy-associated atrial natriuretic factor induction, and c-Jun N-terminal kinase activation by Galphaq, and improved ventricular mechanical function. Thus, MEKK1 mediates cardiac hypertrophy induced by Galphaq in vivo and is a logical target for drug development in heart disease involving this pathway.