The intracompartmental sorting and functional consequences of ectopic expression of the six vertebrate actin isoforms was investigated in different types of cultured cells. In transfected fibroblasts all isoactin species associated with the endogenous microfilament cytoskeleton, even though cytoplasmic actins also showed partial localization to peripheral submembranous sites. Functional and structural studies were performed in neonatal and adult rat cardiomyocytes. All the muscle isoactin constructs sorted preferentially to sarcomeric sites and, to a lesser extent, also to stress-fiber-like structures. The expression of muscle actins did not interfere with cell contractility, and did not disturb the localization of endogenous sarcomeric proteins. In sharp contrast, ectopic expression of the two cytoplasmic actin isoforms resulted in rapid cessation of cellular contractions and induced severe morphological alterations characterized by an exceptional outgrowth of filopodia and cell flattening. Quantitative analysis in neonatal cardiomyocytes indicated that the levels of accumulation of the different isoactins are very similar and cannot be responsible for the observed isoproteins-specific effects. Structural analysis revealed a remodeling of the cytoarchitecture including a specific alteration of sarcomeric organization; proteins constituting the sarcomeric thin filaments relocated to nonmyofibrillar sites while thick filaments and titin remained unaffected. Experiments with chimeric proteins strongly suggest that isoform specific residues in the carboxy-terminal portion of the cytoplasmic actins are responsible for the dominant negative effects on function and morphology.