Previous work showed that voltage-gated Na⁺ channels (VGSCs) are expressed in strongly metastatic human breast cancer (mBCa) MDA-MB-231 cells and that their activity potentiates motility, endocytosis and invasion, involved in the metastatic cascade (Fraser et al. 2002, 2005; Roger et al. 2003). In the present study, we have investigated a possible functional relationship between epidermal growth factor (EGF), well known to be associated with mBCa (e.g. Atalay et al. 2003), and VGSC expression. MDA-MB-231 cells were cultured in serum-free media for 24 h, then seeded into 12 μm-pore transwell plates and treated with EGF (50 or 100 ng/ml) and/or tetrodotoxin (TTX) (0.2 or 10 μM) for 6-8 h. Transverse migration was quatified using tetrazolium MTT assay; lateral motility (over 24 h) was measured by wound-heal assay (Fraser et al. 2005). Each experiment was performed 4-8 times. Data are presented as mean ± S.E.M.; statistical significance was evaluated using Student’s t test. TTX (10 μM) reduced migration by 33 ± 8% (p<0.05; n=8); 0.5 μM TTX had no effect, consistent with underlying potentiation by Nav1.5 activity. An inhibitor of EGF receptor tyrosine kinase, AG1478 (10 μM), reduced motility by 13 ± 2% (p<0.05; n=4), indicating involvement of endogenous EGF in motility. Exogenous EGF significantly increased migration by 44 ± 4% (50 ng/ml) and 73 ± 7% (100 ng/ml) (p< 0.05 for both; n=8). The effect of exogenous EGF was blocked by AG1478 and by co-application with TTX. Thus, EGF (50 ng/ml) + TTX (10 μM) decreased migration by 18 ± 4% (p< 0.05 cf. control; p0.05 cf. TTX-treated; n=8). It is concluded that VGSC activity plays a significant intermediary role in EGF-induced potentiation of metastatic cell behaviour in human BCa in vitro.