Using a panel of monoclonal antibodies to several different cytoskeletal elements in primary cultures derived from newborn rat hearts we report that fibroblasts similar to cardiac-muscle cells expressed the alpha-actin isoform of smooth muscle cells. However, striated muscle alpha-actin or desmin antibodies did not stain cardiac fibroblasts but did stain cardiac-muscle cells. The alpha-smooth muscle actin distributed as a stress fiber and in a cross-striated pattern in cardiac muscle while fibroblasts showed exclusive stress fiber staining. These results suggest that connective tissue cells during development of the heart contain muscle-specific elements which may relate to the organ-specific contractile function with which they are associated.