Background: Controversial results on hyperhomocysteinemia and cardiovascular risk in hemodialysis (HD) could be due in part to the methodology used for homocysteine (Hcy) determination. Objective: The aim of this study was to compare the influence of the method used for Hcy determination (chromatography or immunoassay) with regard to the association of Hcy with cardiovascular mortality rate in HD patients in a 3-year prospective study. Methods: A total of 162 patients undergoing HD were included in a cohort study. Baseline Hcy levels were measured by HPLC and fluorescence polarization immunoassay (FPIA). Cysteine and cysteinylglycine were determined simultaneously with Hcy measurement by HPLC. Results: Hcy levels obtained with both methods were highly correlated (r2=0.927, p<0.0001). An increased relative risk (RR) for cardiovascular mortality (n=31) was found between the highest against lowest tertile of Hcy for both HPLC (RR 2.74, 95% CI 1.07–7.02; p<0.05) and FPIA (RR 2.76, 95% CI 0.99–7.70; p=0.05). Interestingly, increased cysteine (≥452 μmol/L) and cysteinylglycine (≥36.6μmol/L) levels were associated with a decreased RR of non-cardiovascular death (n=26) (RR 0.27, 95% CI 0.09–0.79; p=0.02) for cysteine and (RR 0.28, 95% CI 0.09–0.90; p=0.03) for cysteinylglycine when compared to the first tertile. Conclusions: This study demonstrated an increased risk of cardiovascular mortality in HD patients with Hcy values in the third tertile, independent of the method used. HPLC offers the advantage of simultaneous determination of other aminothiols that appear to be associated with non-cardiovascular mortality. Clin Chem Lab Med 2006;44:949–54.