We describe an immunoassay for thyroxine in serum. In the assay specific antibody covalently bonded to latex particles is used, along with horseradish peroxidase as the label, and o-phenylenediamine as the chromogen. The flexible protocol is designed for manual execution. Performance is similar to that of the highest-sensitivity thyroxine radioimmunoassays. Results correlate well with radioimmunoassay (r = 0.99, slope = 0.93, y-intercept = 2.4 microgram/liter for 201 samples) and an automated enzyme immunoassay (r = 0.97, slope = 0.99, y-intercept = 4.7 coefficients of variation are less than 7.2% over the entire useful range of the assay (20--240 microgram/liter). The limit of detection is less than 94 pg/tube at 20 microgram/liter. Only D-thyroxine is known to interfere with serum assays. This assay has no discernible protein effect from 40 to 80 g of protein per liter, unlike many thyroxine radioimmunoassays. Serum preservatives known to be peroxidase inhibitors do not adversely affect assay performance because of the 56-fold dilution in the final assay mixture. Hemolyzed serum and EDTA-treated plasmas are unsuitable for this assay.