Background/Aims: Nitric oxide (NO) plays a key role in the regulation of vascular tone and controls both local and systemic hemodynamics. Here, we estimated systemic NO production rates of hemodialysis (HD) patients, based on the time course of plasma concentration of nitrate (an oxidative end product of NO) and investigated possible roles of NO-related factors. Methods: We measured plasma concentrations of nitrate, L-arginine (a substrate of NO synthase: NOS), asymmetric dimethylarginine (ADMA, an endogenous NOS inhibitor), tetrahydrobiopterin (BH4, a NOS cofactor), dihydrobiopterin (BH2, an oxidized form of BH4) and oxidized low-density lipoprotein (oxyLDL; an index of oxidative stress) before and after 30-min and 4-hour HD (n = 10). Results: The time-averaged NO production rate during HD was estimated by fitting the time course of plasma nitrate concentration with a single-compartment model (4.00 ± 0.82 µmol/min, 4.99 ± 1.08 µmol/kg/h). The L-arginine/ADMA ratio (L-arginine availability) after 30-min HD showed a positive correlation with the NO production rate (p < 0.05). Conclusion: The systemic NO production rate during HD could be estimated by the single-compartment analysis. The L-arginine/ADMA ratio seems to play an important role in the regulation of the NO production during HD.