We investigated the distribution of α-skeletal, α-cardiac, and α-smooth muscle actin isoforms in human heart during development, hypertrophy, and failure. At 20 weeks of fetal life, α-skeletal actin was localized in a small proportion of subendocardial and papillary muscle cardiomyocytes. At this gestation time, diffuse α-cardiac actin staining was observed, associated with focal expression of α-smooth muscle actin. In normal adult subjects, α-skeletal actin positive cardiomyocytes were distributed in a transmural gradient with the highest proportion located subendocardially. In myocardial hypertrophy and cardiomyopathies, the amount of α-skeletal actin was increased and diffuse staining was seen in all layers of ventricular myocardium, with the exception of idiopathic dilated cardiomyopathies. Cardiomyocytes were negative for α-smooth muscle actin in all pathological situations studied. As expected, fibroblasts in post-infarct scars expressed α-smooth muscle actin and transforming growth factor-β1 but, surprisingly, were negative for these proteins in interstitial fibrosis. Our results demonstrate that increased expression of α-skeletal actin in the diseased human heart is associated with increased myocyte stretch, increased wall stress, and pressure overload, but not with idiopathic dilated cardiomyopathies. They also suggest that fibrotic changes develop with different mechanisms in scars versus interstitial fibrosis. Copyright © 2003 John Wiley & Sons, Ltd.