Expression of nitric oxide synthase (NOS) and voltage-gated Na⁺ channels (VGSCs) have both been linked to progression of prostate cancer (Klotz et al., 1998; Grimes et al., 1995). In the present study, we aimed to answer two questions: (1) Do metastatic prostate cancer cells release nitric oxide (NO). (2) Is VGSC activity involved in this process? Electrochemical detection was used to measure NO release from MAT-LyLu cell line (incubated in Krebs solution) over a period up to 24 hours. This method involved conversion of nitrates and nitrites in solution back to NO by enzymatic reduction (Neal et al., 1998). The specificity of the measurements were tested using two agents: (1) NO scavenger, cPTIO (2-(4-Carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide potassium salt; 0.025, 0.25 and 2.5 μM) and (2) NOS substrate, L-arginine (0.1, 0.5 and 1 μM). The possible effect of the VGSC activity on NO production was tested using tetrodotoxin (TTX; 20 nM and 1 μM). Data are presented as means ± S.E.M. Student’s t-test was used to analyse the data. Under resting conditions, MAT-LyLu cells released NO increasingly with time. There was some variability in the time course of production, but on average, the concentration of NO in solution saturated at a mean value of 8.9 ± 2.2 μM (n = 10) by 9 h. This duration, therefore, was adopted as the sampling period. Incubation with cPTIO (2.5 μM) caused a reduction in the quantity of NO detected by 61 ± 7 % (n = 5; p < 0.05). Conversely, L-arginine (1 μM) induced an increase in NO production by 88 ± 27 % (n = 3; p < 0.05). These effects were dose-dependent. Treatment with TTX (1 μM) increased NO production, on average to 14.8 ± 2.7 μM (n = 10; p < 0.01). Interestingly, however, in a subpopulation (n = 5/10) of the cultures tested, TTX had noticeably little effect. In conclusion, (1) NO is released tonically from strongly metastatic rat prostate cancer cells and (2) VGSC activity, previously shown to be associated specifically with the strongly metastatic phenotype, is involved in the NO release. The available data, taken together, are consistent with NO and VGSC systems being in equilibrium, such that under the experimental conditions used, VGSC activity suppresses NO release. Further work is required to elucidate the mode(s) of VGSC action on NO production and how this relates to metastatic cell behaviour.