BACKGROUND: Biotin causes significant positive interference with Free T4 (thyroxine) measurement using the current Beckman assay. Recently, Beckman reformulated the FT4 assay to overcome biotin interference. We compared the effect of biotin on both current and newly formulated FT4 assays. MATERIALS AND METHODS: Three serum pools were prepared using specimens containing various amounts of FT4. Aliquots of each pool were supplemented with various amounts of biotin. Then FT4 concentrations were measured by both the current and new FT4 assay. In addition, three volunteers ingested 10 mg of biotin. Specimens were drawn prior to the administration of biotin and 2 h post-biotin ingestion. FT4 concentration was measured before and after biotin administration using both assays. RESULTS: The newly formulated FT4 assay correlated well with the current FT4 assay. In the presence of biotin, FT4 concentrations were increased significantly using the current assay. However, negligible changes were observed using the newly formulated assay. We also observed similar results in vivo within the volunteer specimens. CONCLUSION: The newly formulated Beckman Access FT4 assay is free from biotin interference up to a very high biotin concentration of 3500 ng/mL.