We describe a method of isolating and maintaining primary cultures of mouse neonatal cardiac myocytes (NCM). This is derived from the well-established procedure for making NCM cultures from rat neonates by sequential digestion of rat ventricular myocardial pieces using a collagenase/pancreatin mixture. One-day-old mouse neonates are taken and the heart excised. The great vessels, atria, and top section of the ventricular chambers are cut away and the remaining ventricular myocardium is cut into small cubes (about 1–2 mm3 ). Heart pieces from at least 30 animals are then subjected to short (15–25 min) digestion in a shaking water bath in the presence of collagenase and pancreatin. Cell supernatants are taken and pooled together for a total of five digestion steps. The cells are then plated on gelatinized culture dishes and allowed to attach overnight. Myocyte cultures were inspected microscopically for up to 4 days, revealing that many myocytes beat throughout this period. This protocol may be of use for making primary cardiac myocyte cultures from transgenic mice and for investigating gene transcription and cell signalling.