In developmentally regulated D1:S3 splicing of Nav1.5, there are 31 nucleotide differences between the 5′‐exon (‘neonatal’) and the 3′‐exon (‘adult’) forms, resulting in 7 amino acid differences in D1:S3‐S3/S4 linker. In particular, splicing replaces a conserved negative aspartate residue in the ‘adult’ with a positive lysine. Here, ‘neonatal’ and ‘adult’ Nav1.5 α‐subunit splice variants were stably transfected into EBNA‐293 cells and their electrophysiological properties investigated by whole‐cell patch‐clamp recording. Compared with the ‘adult’ isoform, the ‘neonatal’ channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na⁺) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCβ1‐4 mRNA expression levels did not change. The significance of the charge‐reversing aspartate to lysine substitution was investigated by mutating the lysine in the ‘neonatal’ channel back to aspartate. In this ‘neonatal K211D’ mutant, the electrophysiological parameters studied strongly shifted back towards the ‘adult’, that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in ‘neonatal’ Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na⁺ influx. Developmental and pathophysiological consequences of such differences are discussed. J. Cell. Physiol. 216: 716–726, 2008, © 2008 Wiley‐Liss, Inc.