The transmembrane potential (Vm) is established as a functionally instructive biophysical cue in non-excitable cells and has been shown to be a key modulator of cell volume, differentiation, migration and proliferation. Numerous studies have demonstrated a depolarised Vm phenotype as a determinant of cell proliferation across a broad range of cancer cell types (1). The transmembrane potential is generated by ionic gradients and the associated permeability of the membrane to said species. Due to the ubiquitous expression of a vast array of background/leak potassium (K+) channels in normal mammalian cells, K+ is the predominantly permeable ion, and Vm tends towards the reversal potential for K+ (Ek) resulting in a negative membrane potential. The reversal potential for sodium (Na+) is positive and increased plasma membrane permeability to Na+ would result in a more depolarised phenotype. However, the role of Na+ in the contribution to Vm in cancer is still poorly understood. The aim of this study was to systematically investigate the Na+ permeability and the existing Na+ gradient independently in MCF-7 breast and SKOV-3 adenocarcinoma cancer cell lines. Sodium permeability was investigated with the whole-cell current clamp (I=0) technique by replacement of 135 mM Na+ (5mM K)in external physiological salt solution (PSS) with 135mM N-methyl-D-glucamine (NMDG), 5mM K+). The cytosolic Na+ concentration was determined in MCF-7 cells by live cell imaging where we combined a cytosolic pH sensitive dye (5-carboxy-SNARF-1) with a Na+/H+ exchanger to manifest a pH change that is dependent on Na+ gradient. In MCF-7 cells, the Vm in normal PSS was -8.38 ± 4.95 mV (n=5) (mean ± standard error of mean, compared by paired t test). Upon replacement with Na+-free solution, Vm hyperpolarised to -23.5 ± 3.93 mV (n=5) (p<0.05). In SKOV-3 cells Vm in normal PSS was -2.34 mV ± 0.95 (n=9), compared with -15.53 ± 4.65 mV (n=9) (p<0.05) upon replacement with Na+-free solution. The cytosolic Na+ concentration was determined to be 8.43 ± 0.75 mM (n=3) (mean ± standard error of mean). In two distinct cancer cell lines, there exists a persistent permeability to Na+ and thus a contribution to the plasma membrane potential. In MCF-7 cells cytosolic Na+ concentration is comparable to that of non-cancerous non-excitable cells in contrast to values reported using more invasive energy dispersive x-ray microanalysis methods (2). These data indicate that there is a contributory role of Na+ in the establishment of a persistently depolarised Vm in cancer cells, suggesting that sodium dynamics may play a central role in modulating cancer cell proliferation.