The purpose of this study was to compare 2 different nonradioactive assay methods with a conventional radioimmunoassay (RIA) measuring the concentration of serum thyroxine (T4) in horses. Serum was obtained from 85 adult standardbred horses. The T4 concentration of each sample was analyzed by RIA, chemiluminescent enzyme immunoassay (CEI), and homogeneous enzyme immunoassay (HEI). The correlation between the HEI method and RIA method was significantly greater (r = 0.89) than the correlation between the CEI and the reference method (r = 0.53). In addition, the precision of the HEI method was significantly greater than the CEI method; within-run percentage coefficients of variation were 4.5% and 15.9%, respectively, at mean T4 concentrations of 19-20 nmol/liter. On the basis of these findings, the HEI method was evaluated further. Both between-run precision and linearity were deemed adequate upon dilution by the HEI method. In addition, recovery of L-thyroxine added to equine serum was linear over 6 concentrations tested and averaged 79.6% with a manufacturer recommended data correction factor. An in-house correction factor was calculated by linear regression analysis of the RIA and HEI results from the original equine serum samples. Use of this correction factor improved the average recovery to 94.2% while maintaining excellent linearity (r2 = 0.9978). Although both nonradioactive methods of T4 analysis could likely substitute for the RIA reference method, the HEI method had the highest correlation and precision. The HEI technique also yielded adequately accurate results after correction of the data with an appropriate correction factor. Individually derived in-house correction factors may improve the accuracy of the HEI method to a greater extent than manufacturer suggested correction factors.