From mid-gestation, action potentials (APs) are initiated by large transient inward Na+ currents (INa(T)), derived from voltage-gated Na+ channels (VGSCs), primarily Nav1.5 [1]. Nav1.5 can also produce a persistent Na+ current (INa(P)), in which the inactivation component is greatly slowed, which prolongs the cardiac AP and is a major pathway for intracellular Na+ entry [2]. We have found that Nav1.5 has two variants of exon 6 (a 5’ genomic variant and a 3’ variant), which encodes a region of the first transmembrane domain (D1) close to the S4 voltage-sensing segment [3]. This splicing has previously been described for other VGSCs. Splice forms typically only differ at one amino acid (position 7) [3]. The two Nav1.5 variants are unusual in that (i) they differ at 7 amino acids, and (ii) at position 7 of the 5’-variant, the characteristic neutral residue is replaced with a positively-charged lysine. In the original report (of rat Nav1.2 splicing), transcripts with the 5’ variant were abundant at birth but quickly replaced by 3’-variant containing transcripts within days. Thus 5’-variant channels were termed ‘neonatal’ and 3’-, ‘adult’ [4]. We initially found Nav1.5 splicing was similarly developmentally regulated in mouse hearts [2]. However, we did not investigate (a) whether ‘neonatal’ Nav1.5 (nNav1.5) was more abundant than ‘adult’ Nav1.5 (aNav1.5) in neonates, or (b) what changes occur in this splicing during embryonic and foetal development. Here we have used isoform-specific real-time RT-PCRs on mouse hearts from E9.5 to P19.5 to address this. Our data shows that aNav1.5 mRNA levels increase dramatically from E9.5 (~300-fold), peaking at P1.5. In contrast nNav1.5 levels remain relatively constant through to adulthood. nNav1.5 thus contributes proportionally more to cardiac Nav1.5 expression at early embryonic stages than in later development. However, since Nav1.5 at E9.5 is already of critical importance (as E9.5 Nav1.5 -/- mice already display severe cardiac abnormalities), nNav1.5, constituting ~50% of Nav1.5 mRNA at E9.5, is likely to have a major role in heart development. Finally, to determine how Nav1.5 splicing changes relate to excitability changes in the developing heart, nNav1.5 and aNav1.5 channels were stably transfected into EBNA-293 cells and their electrophysiological properties compared. Importantly, differences between the two Nav1.5 isoforms were found that would be consistent with the previously reported loss of spontaneous AP firing and increase in INa(T) kinetics with development [5]. In conclusion, nNav1.5 is expressed at its highest level (proportionally) in the early embryonic heart and its electrophysiological properties are consistent with it making a significant contribution to heart excitability at this stage. The continued expression of this splice variant in adulthood might further suggest it is also important in the adult heart.