A fully automated immunoassay for total plasma homocysteine assay was evaluated at four centers. To measure total homocysteine, oxidized forms of homocysteine in serum and plasma were reduced by dithiothreitol and assayed by a competitive fluorescence polarization technique. The assay had within-run precision from 0.9 to 3.0% and total precision from 2.8 to 4.1% for control materials with homocysteine concentrations of approximately 7, 12.5, and 25 micromol/L, a sensitivity of 0.35 micromol/L, good parallelism upon dilution, and analytical recovery ranging from 97.4 to 103.8%. The immunoassay correlated with four different HPLC assays for homocysteine, yielding a slope of 0.98, an intercept of -0.19 micromol/L, and a correlation coefficient of 0.966 for 440 paired samples. The reference range, determined with plasma samples from 609 males and 600 females, yielded a mean of 9.17+/-2.86 micromol/L, with a central 95% range of 4.78-15.43 micromol/L. The immunoassay is a suitable alternative to HPLC and may be useful in screening persons with high risk of coronary artery disease.