Electrophysiological studies have suggested that strongly metastatic human breast cancer (BCa) cells express functional voltage-gated sodium channels (VGSCs) (Fraser et al. 2002; Roger et al. 2003). In prostate cancer, VGSC activity potentiates a variety of cellular behaviours involved in the metastatic cascade (e.g. Laniado et al. 1997; Mycielska et al. 2003). In fact, it has been suggested that VGSC expression alone is ‘necessary and sufficient’ for cellular invasiveness (Bennet et al. 2003). The aims of this study were to investigate the subcellular localization of VGSC alpha subunit (VGSCα) protein in human BCa cells and its possible functional role. Weakly and strongly metastatic human BCa cell lines (MCF-7 and MDA-MB-231, respectively) were adopted. Western blots and confocal microscopy identified VGSCα protein and its subcellular location (quantified by confocal densitometry). A commercial pan-VGSCα and a novel polyclonal antibody specific for the neonatal Nav1.5 (nNav1.5; expressed predominantly in metastatic BCa) were used. Invasion and proliferation were measured as before (Grimes et al. 1995). Cells were treated with 50 μM forskolin, a stimulator of adenyl cyclase, and/or 10 μM TTX in normal medium for 48 h. Data are presented as mean ± SEM. Statistical analyses were done by Mann-Whitney U test and unpaired t test. Both BCa cell lines expressed VGSCa protein. Subcellular fractionation and confocal microscopy showed that intracellular VGSCα was present, mainly in endoplasmic reticulum and Golgi. However, only MDA-MB-231 cells expressed VGSCαs strongly in plasma membrane (PM). Forskolin had no effect on total VGSCα or nNav1.5 protein in the cells, but increased nNav1.5 expression in PM, by 126 ± 16 % in MCF-7 and 98 ± 21 % in MDA-MB-231 cells (n=37 cells/3 experiments, p <0.05, for both). Forskolin treatment increased invasiveness of MCF-7 and MDA-MB-231 cells by 118 ± 29 % and 120 ± 29 %, respectively (n=9; p<0.05). Co-application of TTX reduced invasiveness to control levels. Regarding proliferation, only forskolin had an effect on MDA-MB-231 cells, reducing it by 16.5 ± 1.7 % (n=8; p<0.001). These results are consistent with forskolin inducing trafficking of intracellular nNav1.5 protein into PM, possibly via protein kinase A activation, and thus increasing the cells' invasiveness.