Background: Controversial results about hyperhomocysteinemia and cardiovascular (CV) risk in hemodialysis (HD) could be due in part to the methodology used for homocysteine (Hcy) determination. Objective: To compare the influence of the method used for Hcy determination (chromatography or immunoassay), in regard to the association of Hcy with CV mortality rate in HD patients in a 3 year prospective study. Methods: One hundred sixty-two patients undergoing HD were included in a cohort study. Baseline Hcy levels were measured by high performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA). Cysteine and cysteinylglycine were determined simultaneously to Hcy measure by HPLC. Results: Hcy levels obtained with both methods were highly correlated (r2=0.927, p<0.0001). An increased relative risk (RR) for CV mortality (n=31) was found between the highest against lowest tertile of Hcy both with HPLC: 2.74 [1.07-7.02, confidence interval 95% (CI)] (p<0.05) and with FPIA: 2.76 [0.99-7.70, CI 95%] (p=0.05). Interestingly, increased cysteine (≥452 µmol/L) and cysteinylglycine (≥36.6 µmol/L) levels were associated with decreased RR of non-CV death (n=26) (RR: 0.27 [0.09-0.79, CI 95%], p=0.02) for cysteine and RR: 0.28 [0.09-0.90, CI 95%], p=0.03 for cysteinylglycine when compared versus first tertile. Conclusion: This study demonstrated an increased risk of CV mortality in HD patients with Hcy values in the third tertile, independently to the method used. HPLC offers the advantage of simultaneously determination of other aminothiols that appear associated with non-CV mortality.