Damage to the cardiac myocyte sarcolemma following any of several pathological insults such as ischemia (anoxia) alone or followed by reperfusion (reoxygenation), is most apparent as progressive sarcolemmal blebbing, an event attributed by many investigators to a disruption in the underlying cytoskeletal scaffolding. Scanning electron microscopic observation of tissue cultured rat neonatal cardiomyocytes indicates that exposure of these cells to the toxic aldehyde 4-hydroxynonenal (4-HNE), a free radical-induced, lipid peroxidation product, results in the appearance of sarcolemmal blebs, whose ultimate rupture leads to cell death. Indirect immunofluorescent localization of a number of cytoskeletal components following exposure to 4-HNE reveals damage to several, but not all, key cytoskeletal elements, most notably microtubules, vinculin-containing costameres, and intermediate filaments. The exact mechanism underlying the selective disruption of these proteins cannot be ascertained at this time. Colocalization of actin indicated that whereas elements of the cytoskeleton were disrupted by increasing length of exposure to 4-HNE, neither the striated appearance of the myofibrils nor the lateral register of neighboring myofibrils was altered. Monitoring systolic and diastolic levels of intracellular calcium ([Ca2+]i) indicated that increases in [Ca2+]i occurred after considerable cytoskeletal changes had already taken place, suggesting that damage to the cytoskeleton, at least in early phases of exposure to 4-HNE, does not involve Ca(2+)-dependent proteases. However, 4-HNE-induced cytoskeletal alterations coincide with the appearance of, and therefore suggest linkage to, sarcolemmal blebs in cardiac myocytes. Although free radicals produced by reperfusion or reoxygenation of ischemic tissue have been implicated in cellular damage, these studies represent the first evidence linking cardiomyocyte sarcolemmal damage to cytoskeletal disruption produced by a free radical product.