RT-PCR shows that mouse skeletal muscle contains type-2 iodothyronine deiodinase (D2) mRNA. However, the D2 activity has been hard to measure. Except for newborn mice, muscle homogenates have no detectable activity. However, we have reported D2 activity in mouse muscle microsomes. As the mRNA, activity is higher in slow-than in fast-twitch muscle. We addressed here the major problems in measuring D2 activity in muscle by: homogenizing muscle in high salt to improve yield of membranous structures; separating postmitochondrial supernatant between 38 and 50% sucrose, to eliminate lighter membranes lacking D2; washing these with 0.1 M Na 2 CO 3 to eliminate additional contaminating proteins; pretreating all buffers with Chelex, to eliminate catalytic metals; and eliminating the EDTA from the assay, as this can bind iron that enhances dithiothreitol oxidation and promotes peroxidation reactions. Maximum velocity of T 3 generation by postgradient microsomes from red muscles was approximately 1100 fmol/(h ⅐ mg) protein with a MichaelisMenten constant for T 4 of 1.5 nM. D2-specific activity of Na 2 CO 3 -washed microsomes was 6 -10 times higher. The enrichment in D2 activity increased in parallel with the capacity of microsomes to load (sarco/endoplasmic reticulum Ca 2ϩ -ATPase) and bind Ca 2ϩ (calsequestrin), indicating that D2 resides in the inner sarcoplasmic reticulum, close to the nuclei. The presence of D3 in the sarcolemma suggests that the most of D2-generated T 3 acts locally. Estimates from maximum velocity, MichaelisMenten constant, and muscle T 4 content suggest that mouse red, type-1, aerobic mouse muscle fibers can generate physiologically relevant amounts of T 3 and, further, that muscle D2 plays an important role in thyroid hormone-dependent muscle thermogenesis. (Endocrinology 152: 3082-3092, 2011) T he main product of thyroid secretion is T 4 , which has weak hormonal activity. The enzyme-catalyzed removal of the 5Ј iodine of T 4 generates T 3 , which has at least 10 times more activity and a much faster effect, being considered the active form of the hormone, ultimately responsible for virtually all the effect of the thyroidal secretion. There are two 5Јiodothyronine deiodinases, type-1 iodothyronine deiodinase and type-2 iodothyronine deiodinase (D2). A third deiodinase, called type-3 iodothyronine deiodinase (D3) removes the 5 iodine, from the inner ring of T 4 or T 3 , generating, respectively, rT 3 and 3,3Ј-T 2 , both devoid of biological activity. These three enzymes have not only an important role in modulating circulating T 3 levels, but D2 and D3 are particularly important in regulating the intracellular concentration of T 3 in a tissuespecific manner (see Refs. 1, 2 for reviews). This provides an additional and physiologically critical level of regulation that allows tissues to vary the level of intracellular T 3 according to specific needs, for example, in stages of central nervous system development (3), regenerating liver (4) and skeletal muscle (5), regionally in the ischemic or hypertrop...