Cloning of the individual regulatory (R) and catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) and expression of these subunits in cell culture have provided mechanistic answers about the rules for PKA holoenzyme assembly. One of the central findings of these studies is the essential role of the RI alpha regulatory subunit in maintaining the catalytic subunit under cAMP control. The role of RI alpha as the key compensatory regulatory subunit in this enzyme family was confirmed by gene knockouts of the three other regulatory subunits in mice. In each case, RI alpha has demonstrated the capacity for significant compensatory regulation of PKA activity in tissues where the other regulatory subunits are expressed, including brain, brown and white adipose tissue, skeletal muscle, and sperm. The essential requirement of the RI alpha regulatory subunit in maintaining cAMP control of PKA activity was further corroborated by the knockout of RI alpha in mice, which results in early embryonic lethality due to failed cardiac morphogenesis. Closer examination of RI alpha knockout embryos at even earlier stages of development revealed profound deficits in the morphogenesis of the mesodermal embryonic germ layer, which gives rise to essential structures including the embryonic heart tube. Failure of the mesodermal germ layer in RI alpha knockout embryos can be rescued by crossing RI alpha knockout mice to C alpha knockout mice, supporting the conclusion that inappropriately regulated PKA catalytic subunit activity is responsible for the phenotype. Isolation of primary embryonic fibroblasts from RI alpha knockout embryos reveals profound alterations in the actin-based cytoskeleton, which may account for the failure in mesoderm morphogenesis at gastrulation.