Transmural gradients in action potential duration (APD) and Ca²⁺ handling proteins are important for both the normal functioning of the ventricle and arrhythmogenesis. In the rabbit, the transmural gradient in APD is minimal in the neonate. During post-natal development, APD increases both in the epicardium and the endocardium, but the prolongation is more substantial in the endocardium leading to a significant transmural gradient. We have investigated changes in ion channel expression in the subepicardial and subendocardial layers of the left ventricular free wall in neonatal (2-7 days of age; n=11) and adult male (~6 months of age; n=11) New Zealand White rabbits using quantitative PCR (qPCR), in situ hybridisation (ISH) and immunohistochemistry. The rabbits were killed humanely in accordance with the regulations of the United Kingdom Animals (Scientific Procedures) Act 1986. qPCR revealed that in the neonate, Nav1.5 (responsible for I_Na), ERG (responsible for I_K,r) and minK (responsible for I_K,s) mRNAs were more abundant in the endocardium than the epicardium, whereas the reverse was true for KChIP2 (in part responsible for I_to) mRNA. Moreover, in the adult, Cav1.2 (responsible for I_Ca,L), SERCA2a, RyR2, ERG, KvLQT1 (responsible for I_K,s) and KChIP2 mRNAs were more abundant in the epicardium than the endocardium. Consistent with this, ISH showed that KChIP2 mRNA was more abundant in the epicardium than the endocardium both in the neonate and adult. However, in the neonate, whereas Nav1.5 mRNA was more abundant in the endocardium than the epicardium, it was uniformly distributed across the ventricle in adult. Cav1.2 mRNA was uniformly distributed across the neonatal ventricle, while in the adult ventricle, Cav1.2 mRNA was significantly more abundant in the epicardium than the endocardium. Immunohistochemistry confirmed that NCX1, SERCA2a and RyR2 proteins were more abundant in the epicardium than the endocardium in the adult, but not in the neonate. To conclude, there are complex developmental changes in ion channel and Ca²⁺ handling protein expression across the ventricle that may have implications for the treatment of arrhythmias.