The neonatal heart is a very useful tool for the study of biochemical pathways and properties of cardiomyocytes and as it has the potential to regenerate for a brief period of time from birth; it is also useful to study cardiac regeneration. However, as the heart matures, this proficiency for regeneration is reduced. This regenerative potential may be influenced by the microenvironment of the heart in the early stages of postnatal development and therefore, cell cultures derived at this stage may contain functional cardiomyocytes and progenitor cells. The aim of this study was to identify key steps in the isolation and culture of such early stage-neonatal mouse hearts to allow maximum migration of cardiomyocytes from the explant and their maintenance as functional, long term cultures. Explant cultures of mouse ventricles preserved 3-dimensional structure and generated migrating layers of cardiomyocytes that expressed alpha sarcomeric actin which could be further sub-cultured by enzymatic dissociation. Western blotting demonstrated expression of c-KIT, GATA4, alpha sarcomeric actin and connexin43 proteins after 20 days of explant culture. ACTA1, GATA4, and CX43 continued to express in five weeks old explant cultures while the c-KIT protein was expressed up to two passages during sub-culture. Real time PCR and SQRT PCR also demonstrated gene expression of cardiomyocyte markers in long term cultures. Migrating cells from the explants assembled into contracting spheroids after subculture and expressed the c-KIT protein. Progenitor markers CD44, CD90, and extracellular proteins, periostin and vimentin demonstrated the preservation of cellular heterogeneity in such cultures. Supplementation with Hydrocortisone maintained a cardioprotective environment and reduced the non-myocyte population. This is an optimized and efficient method for the generation of neonatal heart cultures that is not labor intensive and does not require supplementation with cytokines. © 2015 Elsevier GmbH.