Previous studies have suggested that the GATA4 transcription factor plays an important role in regulating mammalian cardiac development. In the studies described in this report we have used gene targeting to produce GATA4-deficient mice. Homozygous GATA4-deficient (GATA4-/-) mice died between 8.5 and 10.5 days post coitum (dpc). GATA4-/- embryos displayed severe defects in both rostral-to-caudal and lateral-to-ventral folding, which were reflected in a generalized disruption of the ventral body pattern. This resulted in the defective formation of an organized foregut and anterior intestinal pore, the failure to close both the amniotic cavity and yolk sac, and the uniform lack of a ventral pericardial cavity and heart tube. Analysis of cardiac development in the GATA4-/- mice demonstrated that these embryos developed splanchnic mesoderm, which differentiated into primitive cardiac myocytes that expressed contractile proteins. However, consistent with the observed defect in ventral morphogenesis, these GATA4-/- procardiomyocytes failed to migrate to the ventral midline to form a linear heart tube and instead formed aberrant cardiac structures in the anterior and dorsolateral regions of the embryo. The defect in ventral migration of the GATA4-/- procardiomyocytes was not cell intrinsic because GATA4-/- cardiac myocytes and endocardial cells populated the hearts of GATA4-/- -C57BL/6 chimeric mice. Taken together, these results demonstrated that GATA4 is not essential for the specification of the cardiac cell lineages. However, they define a critical role for GATA4 in regulating the rostral-to-caudal and lateral-to-ventral folding of the embryo that is needed for normal cardiac morphogenesis.