During metastasis, cancer cells experience different substrates and undergo adhesion/detachement. We have shown previously that strongly metastatic prostate cancer (PCa) cells express voltage-gated Na channels (VGSCs) (e.g. Grimes et al.,1995). Blocking VGSCs with tetrodotoxin (TTX) suppresses cellular behaviours integral to the metastatic cascade, e.g. motility (Fraser et al.,2003) and secretory membrane activity (Mycielska et al.,2003). Here, we questioned whether VGSC activity could also be involved in cellular adhesion. The strongly metastatic rat PCa Mat-LyLu cells were grown on plain glass (PG) or gelatine-coated glass (GG) coverslips for up to 48 h with drugs added 3 h after plating. Voltage-gated currents were determined by whole-cell patch clamp recording. The holding potential was -100 mV. Maximal VGSC current densities were measured and analysed as means sem (n>20) and Student’s t-test. VGSC expression was also studied by immunocyochemistry and confocal microscopy. Mat-LyLu cells grown on PG had VGSC current densities of 23.5 3.0 nA/pF (24 h) and 23.7 2.7 nA/pF (48 h). These were lower for cells grown on GG: 19.5 2.1 nA/pF (24 h) and 15.7 1.9 nA/pF (48 h). Resting membrane potential were depolarized (p<0.001): -48 2 mV (PG) and -41 2 mV (GG). Pre-incubation of cells grown on PG with TTX (1 uM) resulted in no change in current density (p=026). In contrast, Mat-LyLu cells grown on GG for 48 h in equimolar TTX had a significantly higher VGSC current density: 21.8 3.6 nA/pF (p<0.02 cf. untreated). Aconitine (100 uM), a VGSC ‘opener’ (Fraser et al.,2003) applied similarly had the opposite effect, giving the following VGSC current densities: 10.1 2.3 nA/pF (PG) and 3.7 0.6 nA/pF (GG). Similar changes were seen by immunocytochemistry. In conclusion, (1) substrate does influence VGSC expression/activity in Mat-LyLu cells; (2) forcing the cells to adhere more strongly (GG vs. PG) down-regulates VGSC expression; and (3) this change is controlled by VGSC activity. This study provides further evidence that VGSC upregulation could play a significant role in PCa metastasis directly by enhancing cellular activity as well as indirectly by modulating cellular signalling (e.g. Brackenbury & Djamgoz, 2003).