The pathogenesis of heart disease and the development of myocardial failure are highly dependent upon the cardiomyocyte—the basic contractile cell within the heart. Understanding and elucidating the complex networks that regulate cardiomyocyte function are central to the development of specific target-based therapeutic interventions. The relative recent advances in molecular genetics and generation and routine usage of transgenic and knockout mouse models have further necessitated that previously established cardiomyocyte methods be adapted for the isolation, culture, and study of primary adult murine cardiomyocytes, both freshly isolated and in culture. Such adaptation is based not only upon scalability of established techniques, as the mouse heart is an order of magnitude smaller than the rat heart, but also upon properties unique to the mouse. This chapter therefore describes current methods for the isolation, culture, and functional analysis of adult murine cardiomyocytes.