Type I iodothyronine deiodinase (ID-I) activity is impaired in C3H/He (C3H) mice compared with BALB/c and C57BL/6N (C57) mice. In this study we compared ID-I activity and protein labeling with N-bromoacetyl(-)[125I]T3 (BrAc[125I]T3) or 75Se in liver microsomes of C3H and C57 mice. Hepatic ID-I activity in C3H mice was highly variable with a median of only 18% of that in C57 mice. However, C3H mice had normal serum T4 and T3 levels, although serum reverse T3 was increased. The 28-kilodalton (kDa) ID-I protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of BrAc[125I]T3-labeled microsomes. Labeling of this protein was virtually undetectable in C3H samples with low enzyme activity. ID-I activity in liver microsomes was strongly decreased in Se-deficient mice, which was paralleled by a drastic decrease in BrAc[125I]T3-labeling of the 28-kDa band compared with control mice. Labeling of ID-I with 75Se was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of liver microsomes of [75Se]selenite-injected mice. 75Se labeling of the 28-kDa band was markedly higher in Se-deficient than in control mice and was also markedly higher in C57 than in C3H mice. Finally, liver ID-I messenger RNA (mRNA) was measured on Northern blots using a rat ID-I complementary DNA probe. Messenger RNA levels correlated strongly with ID-I activity, showing a significant decrease in C3H mice. We conclude that in mice, like in rats and humans, ID-I is a selenoprotein. ID-I activity is impaired in C3H mice because of decreased transcription of the ID-I gene or reduced stability of the mRNA.