We recently identified a ‘neonatal’ splice variant of Nav1.5 (nNav1.5) in metastatic human breast cancer (BCa) cells (Fraser et al. 2005). The splicing results from the alternate inclusion of two exons (the 5’ genomic and the 3’ genomic) encoding D1:S3 and the D1:S3/S4 extracellular linker of the voltage-gated Na⁺ channel (VGSC). The ‘neonatal’ (5’ exon) splice variant has 31 nucleotide differences from the ‘adult’ (3’ exon) form (aNav1.5) that results in 7 amino acid substitutions. Of particular note is the replacement of the 3’ exon aspartate with a positively charged lysine residue (Chioni et al. 2005). The aim of the present study was to investigate the possible electrophysiological consequences of the Nav1.5 D1:S3 splicing. All data are presented as mean ± standard errors of the mean. Statistical significance was determined by unpaired t test (n>16 cells for each test). Whole-cell patch clamp recording from transfected EBNA-293 cell lines (Chioni et al. 2005) revealed that aNav1.5 and nNav1.5 activated at -59.7 ± 0.8 mV and -57.8 ± 0.9 mV (P = 0.12) and peaked at -20.3 ± 1.0 mV and -14.2 ± 2.1 (P < 0.01), respectively. The normalized conductance-voltage relationship fitted to a Boltzmann distribution indicated half-activation voltages of -40.6 ± 0.2 mV and -33.8 ± 0.2 mV for aNav1.5 and nNav1.5 (P < 0.001) whilst the slope factors were 6.2 ± 0.2 mV and 7.1 ± 0.2 mV(P < 0.01), respectively. In addition, the half-inactivation voltages for aNav1.5 and nNav1.5 were -94.5 ± 0.4 and -96.0 ± 0.4 mV (P < 0.01), whilst the slope factors were -6.8 ± 0.4 mV and -7.3 ± 0.3 mV (P = 0.43), respectively. The ‘adult’ and ‘neonatal’ Nav1.5 VGSCs differed notably in their kinetics of activation and inactivation. For both splice variants, time to peak decreased with increasing depolarization, from 1.41 ± 0.06 ms at -40 mV to 0.67 ± 0.02 ms at 0 mV (for aNav1.5) and from 1.82 ± 0.07 ms at -40 mV to 0.78 ± 0.03 ms at 0 mV (for nNav1.5). Over the voltage range -30 mV to 0 mV, nNav1.5 activated significantly more slowly than the ‘adult’. The fast and slow inactivation time constants were significantly smaller for aNav1.5 vs nNav1.5 for most voltages analyzed. On the other hand, the time courses of recovery from inactivation were the same at given voltages. In conclusion, significant differences were found in the electrophysiological characteristics of the ‘adult’ and ‘neonatal’ splice variants of Nav1.5 in vitro. Further understanding of such properties in vivo would be of importance for developmental physiology and clinical management of BCa (Fraser et al. 2005).