Advances in our understanding of cardiomyocyte cell biology have been dependent largely upon the ability to generate primary cultures from enzymatically dispersed fetal, neonatal, or adult hearts. Primary cardiomyocyte cultures recapitulate many of the physiologic and molecular attributes found in intact hearts at the corresponding developmental stage. Moreover, these cultures are readily amenable to a wide variety of physical, physiologic, and molecular analyses. Gene transfer approaches including traditional calcium phosphate and lipofection techniques, as well as viral transduction with recombinant retro-, adeno-, or adeno-associated viruses are also readily accomplished. In light of these attributes, primary cardiomyocyte cultures constitute an extremely versatile experimental system.