Technical Abstract: The thyroid hormones function in regulation of vertebrate metabolism, growth, skeletal maturation, and development of the nervous system. Conversion of thyroxine to the metabolically active form triiodothyronine (T3) occurs primarily in the liver by type I 5'-deiodinase (DIO1). A primer set was designed to amplify putative intron 2 of the bovine DIO1 gene. Four single nucleotide polymorphisms were detected by sequence analysis of PCR amplicons in sires of the U.S. Meat Animal Research Center cattle reference population. The bovine DIO1 was mapped by two-point analysis to chromosome 3 (BTA3; http://www.marc.usda.gov/genome/) nearest the marker BM7225 (LOD = 14.94; recombination fraction = 0.03). The assignment of DIO1 to BTA3 is consistent with the predicted location based on the current human-bovine comparative map (http://bos.cvm.tamu.edu/htmls/rhbov3.html). Quantitative trait loci associated with clinical mastitis in dairy cattle and milk protein percentage have been identified in this region of BTA3. Due to the importance of T3 in milk synthesis and energy metabolism, DIO1 is a positional candidate gene contributing to these traits. Accession Nos. BV005735-BV005736