Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 ± 0.5 × 10⁵ cells/left ventricle, 83.8 ± 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2–3 days. The high percentage of viable myocytes after 1 day in culture (72.5 ± 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult β₁/β₂-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either β₁- or β₂-AR, which occurred in 100% of cells, rescued the functional response to β-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.