Cardiovascular disease (CVD) is the leading cause of death in the United States. Elevations in homocysteine (Hcy) have been associated with increased risk of acute coronary syndrome, stroke and peripheral vascular disease. Increased utilization of Hcy as a risk marker has prompted the need for high throughput methods that are simple to use and analytically accurate and precise. We report the performance characteristics of the automated Bayer ADVIA Centaur chemiluminescent Hcy assay. Centaur Hcy is based on a three-step procedure: (1) reduction of Hcy disulfides to free Hcy, (2) enzymatic conversion of free Hcy to S-adenosyl Hcy (SAH) and (3) quantitation of SAH in a competitive immunoassay (labeled anti-SAH antibody: magnetic particles coupled with SAH). Total assay precision ranged from 3.5% to 6.8% at 4.9-62 micromol/Hcy; linearity undiluted from 0 to 65 micromol/l, up to 650 micromol/l with automatic dilution. Method comparisons with fluorescent polarization immunoassay and high-performance liquid chromatography (HPLC) gave linear regression equations with slopes between 0.95 and 1.0. Measurement of Hcy concentrations in apparently healthy populations yielded middle 95th percentile of 9.7 micromol/l, consistent with epidemiologic studies suggesting that 9-10 micromol/l represents the lower threshold of a population at risk of CVD. The Centaur Hcy assay is a sensitive and precise assay for the measurement of Hcy.