The effect of thiamine (vitamin B(1)) or riboflavin (vitamin B(2)) availability on fasting total homocysteine (tHcy) plasma levels in end-stage renal disease patients is unknown. A cross-sectional study was performed in a population of non-vitamin supplemented patients maintained on continuous ambulatory peritoneal dialysis. Red blood cell availability of thiamine (alpha-ETK) and of riboflavin (alpha-EGR), along with other predictors of tHcy plasma levels, was considered in the analysis. There was a linear association of alpha-EGR with tHcy plasma concentrations (P = 0.009), which was not observed for alpha-ETK. Among red blood cell vitamins, alpha-EGR was the only predictor of tHcy levels (P = 0.035), whereas alpha-ETK, red blood cell pyridoxal-5-phosphate supply (alpha-EGOT) and red blood cell folate levels had no effect. The risk for having a high tHcy plasma levels within the fourth quartile (plasma tHcy >38.3 micromol/L) was increased by an alpha-EGR > median (odds ratio, 4.706; 95% confidence interval, 1.124 to 19.704; P = 0.026). By way of contrast, alpha-ETK had no effect in these analyses. Independent predictors of tHcy plasma levels were serum albumin, alpha-EGR, red blood cell folate, and certain MTHFR genotypes. A logistic regression analysis showed that the MTHFR genotype is a predictor for having a tHcy plasma concentration within the fourth quartile. In summary, riboflavin availability, as measured by alpha-EGR, is a determinant of fasting tHcy plasma levels in peritoneal dialysis patients. This finding may have implications for tHcy lowering therapy in individuals with end-stage renal disease.