Steroid receptor coactivator-1 (SRC-1) is a transcription cofactor that enhances the hormone-dependent action mediated by the thyroid hormone (TH) receptor (TR) as well as other nuclear receptors. However, it is not known whether the SRC-1-mediated activation of TH-regulated gene transcription is TR isoform specific in the pituitary. We generated mice that were deficient in TRα and SRC-1 (TRα^0/0SRC-1^−/−), as well in TRβ and SRC-1 (TRβ^−/−SRC-1^−/−), and thyroid function tests and effects of TH deprivation and TH treatment were compared with wild-type mice or mice with deletion of either TRs or SRC-1 alone. We have shown that 1) TRβ^−/−SRC-1^−/− mice demonstrate more severe TH resistance than either the SRC-1^−/− or TRβ^−/− mice; the additive effect indicates that SRC-1 has an independent role in TH action over that of TRβ; 2) SRC-1 facilitates TRβ and TRα-mediated down-regulation of TSH, as TRα^0/0SRC-1^−/− mice demonstrate TH resistance rather than hypersensitivity as seen in TRα^0/0mice; and 3) a compensatory increase in SRC-1 expression is associated with the TH hypersensitivity seen in TRα-deficient animals. We conclude that SRC-1 action in the pituitary mediates TH action via specific TR subtypes.