A fluorescence polarization immunoassay (FPIA) has been commercially released for routine large-scale testing of total homocysteine (tHcy) on the AxSYM analyzer. We evaluated the analytical performance of the AxSYM tHcy FPIA and compared it with the well established high-performance liquid chromatography (HPLC) and IMx tHcy FPIA methods. Homocysteine concentrations were measured by AxSYM and IMx tHcy FPIA and by a rapid isocratic HPLC method with fluorescence detection. Coefficient of variation (CV) of total imprecision for AxSYM tHcy was < or = 5%, mean dilution recovery 102%, analytical sensitivity 0.70 micromol/l and linearity was good up to 1:8 dilution. Spearman rank correlations, rho, were 0.83 (p < 0.0001) for AxSYM vs. HPLC, 0.97 (p < 0.0001) for AxSYM vs. IMx and 0.83 (p < 0.0001) for IMx vs. HPLC. Passing and Bablok regression Y-intercepts and slopes were: 2.944/0.937 (AxSYM vs. HPLC), -0.367/ 1.142 (AxSYM vs. IMx) and 2.632/0.805 (IMx vs. HPLC). Corresponding mean differences (AxSYM-Comparison Assay) recorded over a 5-50 micromol/l measured range were 1.80, -0.73 and 2.53 micromol/l. AxSYM tHcy FPIA's first rate precision, supported by the complete automation of the AxSYM analyzer, makes it fit for routine use and suitable for laboratories requiring homocysteine high-throughput testing capabilities.