alpha-Actinin is an actin binding protein that assists in the stabilization of the plasma membrane and helps to fix organelles in position in a variety of cell types. In muscle, it is a major component of the Z-lines of organized myofibrils. Ankyrin binds to various elements of the cytoskeletal system including microtubules, microfilaments, and intermediate filaments and may help to anchor these structures to the cell membrane. Filamin is a well-characterized actin-associated protein first isolated from chicken smooth muscle. In addition, filamin is a gel-forming protein which aids in the formation of a loose, yet thick, network of actin filaments. These proteins work together, in conjunction with other cytoskeletal proteins, to permit the contractions of heart muscle cells in vertebrates. In a unique strain of the axolotls (Ambystoma mexicanum) a simple recessive mutation, designated by gene c, results in an incomplete differentiation of the hearts of affected embryos. Although the mutant (c/c) embryos form hearts, they do not beat because of a failure in the formation of organized sarcomeric myofibrils. The current study was undertaken to examine the three-dimensional distributions of three different contractile-cytoskeletal proteins (alpha-actinin, ankyrin, and filamin) during myofibrillogenesis in normal and mutant hearts from early heart-beat stage 37 through advanced embryonic stage 42. Our results demonstrate that the contractile proteins become increasingly better organized in normal hearts as development progresses. In mutant hearts, although the proteins are present in almost normal amounts, they fail to form normally organized myofibrils.