Patients undergoing hemodialysis have impaired metabolism of such sulfur-containing amino acids as cysteine (Cys) and homocysteine (Hcy), which may lead to accelerated atherosclerosis. Considering that Cys is mainly synthesized from Hcy, a common C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene may affect the serum total Cys (tCys) concentration, as well as total Hcy (tHcy) concentration, through reduced remethylation of Hcy to methionine, even in hemodialysis patients. To identify the independent determinants for the tCys concentration in dialysis patients, we determined MTHFR C/T genotypes and serum concentrations of tHcy, tCys, and vitamins as cofactors in 464 hemodialysis patients. Serum tCys concentration was positively associated with serum tHcy concentration and negatively associated with the MTHFR mutation, although the mutation correlated positively with serum tHcy concentration. Slopes of regression lines relating tHcy and tCys concentrations differed between the MTHFR genotypes, and the relationship was strengthened with a decreasing number of T alleles. Additionally, serum concentrations of folate and vitamin B(12) correlated positively with tCys concentration, whereas they correlated negatively with tHcy concentration. These findings suggest that the MTHFR mutation is an independent predictor for serum tCys concentrations in hemodialysis patients and that a tCys-decreasing effect of the mutation may arise largely from its attenuation of the positive Cys-Hcy correlation. The tCys-increasing effect of folate and vitamin B(12) appears to be linked to their enhancement of Hcy remethylation.