Cardiomyocyte DNA synthesis was examined in normal and injured adult mouse hearts. In preliminary studied DNA synthesis was monitored by [3H]thymidine incorporation, followed by autoradiographic analysis of dispersed cell preparations. No synthetic cells were identified when 20,000 ventricular cardiomyocytes from normal adult hearts were examined. A high throughput assay was developed to establish the actual labeling index for the adult mouse heart. The assay utilized [3H]thymidine incorporation in transgenic mice which expressed a nuclear-localized beta-galactosidase (beta-Gal) reporter gene exclusively in cardiac myocytes. Cardiomyocyte DNA synthesis was evidenced by colocalization of beta-Gal activity and silver grains in autoradiograms of histological sections. Examination of 180,000 ventricular cardiomyocyte nuclei from normal adult transgenic mice identified a single synthetic nucleus, suggesting a maximum labeling index of 0.0005%. Cardiomyocyte DNA synthesis was next examined in hearts injured by focal cauterization of the left ventricular free wall. Only three synthetic nuclei were identified when 36,000 cardiomyocyte nuclei in the perinecrotic zone of the injured heart were examined. No additional synthetic nuclei were identified when 180,000 nuclei in regions distal to the necrotic zone were examined. These data confirm that cardiomyocyte DNA synthesis in the adult mouse heart is extremely rare and provide baseline data for analyses in genetically modified animals.