It has been shown previously that functional voltage-gated Na+ channels (VGSCs) are expressed in strongly metastatic rat (MAT-LyLu) and human (PC-3) prostate cancer cells and can potentiate invasion in vitro (Grimes et al. 1995; Laniado et al. 1997; Bennett et al. 2004). However, the mechanism(s) controlling VGSC expression in these cells is not known. We reported earlier that long-term (> 24 h) exposure to tetrodotoxin (TTX) can suppress VGSC activity in MAT-LyLu cells (Brackenbury and Djamgoz, 2003). In the present study, we have investigated whether protein kinase A (PKA) is also involved in VGSC regulation. MAT-LyLu cells were pre-incubated with a PKA inhibitor (KT5720) for 48 h, starting 3 h after seeding. Expression of Nav1.7 mRNA, predominant in these cells (Diss et al. 2001), was quantified using real-time PCR and normalised to rat cytochrome b5 reductase (Livak and Schmittgen, 2001). Functional VGSC activity was assayed by whole-cell patch clamp recording (pipette Cs+ used to eliminate K+ currents). Data are presented as mean ± SEM. Statistical significance was evaluated with Student’s t tests. Pre-treatment with KT5720 (500 nM) significantly reduced Nav1.7 expression by 45 ± 0.17 % (P < 0.05; n = 3). A lower (50 nM) concentration had no effect (P = 0.47; n = 3). Pre-treatment with TTX (1 μM) resulted in a similar reduction of 52 ± 0.12 % (P < 0.01; n = 3). Co-application of KT5720 (500 nM) with TTX (1 μM) reduced Nav1.7 expression by 58 ± 0.16 % (P <0.01; n = 3), which was not significantly different to the reduction caused by KT5720 alone (P = 0.30; n = 3). Pre-treatment with KT5720 (500 nM) significantly reduced peak VGSC current density from 14.8 ± 1.7 pA/pF to 4.7 ± 0.7 pA/pF (P < 0.001; n = 11-15). A lower concentration (50 nM) of KT5720 again had no effect (P = 0.24; n = 14-15). The results indicate that inhibition of PKA can reduce functional VGSC activity in MAT-LyLu cells, and at least a part of this regulation is transcriptional. Furthermore, these results are consistent with PKA being an intermediary component of the activity-dependent VGSC regulation in MAT-LyLu cells (Brackenbury and Djamgoz, 2003). Accordingly, from the available evidence taken together, we propose the following scheme: VGSC↑ → Na+ → PKA → VGSC↑.