mRNA for a thyroid hormone receptor isoform that is unique to the pituitary gland (TRβ-2) is down-regulated by T₃. Increases in the expression of this mRNA are seen in rats rendered hypothyroid by treatment with propylthiouracil (PTU). This study used dual labeling to determine which pituitary cells expressed TRβ-2 mRNA in normal and PTU-treated rats. In situ hybridization protocols localized the mRNA (with biotinylated complementary oligonucleotide probes detected by avidin-biotin-peroxidase), and immunoperoxidase protocols identified the pituitary hormone proteins. In dispersed pituitary cells, 20 ± 2% (average ± SD) of cells from normal rats and 30 ± 3% of cells from PTU-treated rats were labeled for TRβ-2 mRNA. PTU caused increases in the area of the labeled cells (from 114 ± 11 to 225 ± 7 μm²), the area of the label per cell (from 27 ± 3 to 71 ± 11 μ²), and label density. PTU produced increases in the percentage of TSH cells from 8 ± 1% to 19 ± 2%, decreases in the percentage of GH cells from 27 ± 3% to 11 ± 2%, and no change in other cell types. After dual labeling, 73% of cells that expressed TRβ-2 mRNA stored either TSH (35 ± 8) or GH (38 ± 6). Less than 10% stored other hormones. When each cell type was analyzed, 56 ± 3% of TSH cells and 43 ± 4% of GH cells expressed TRβ-2 mRNA. When these percentages were multiplied by the percentages of each cell type in the overall population, TSH and GH cells with TRβ-2 mRNA represented 6.8 ± 1 % and 11.6 ± 1% of the pituitary cells, respectively. Less than 1% of all pituitary cells expressed TRβ-2 and ACTH (0.9 ± 0.06), LH (0.8 ± 0.1), FSH (0.8 ± 0.1), and PRL (0.9 ± 0.04). PTU treatment increased the percentage of TSH cells with TRβ-2 mRNA to 72 ± 4% and decreased the percentage of GH cells with TRβ-2 mRNA to 30 ± 3%. However, some enlarged putative TSH cells could not be identified by immunolabel because the storage levels were low. Thus, changes in TRβ-2 mRNA in hypothyroid rats may be the net result of the increase in the percentage of TSH cells, the amount of mRNA per cell (measured by area and density of label), and the decrease in the percentage of GH cells. The divergent effects of the removal of T₃ on expression of this mRNA by TSH and GH cells indicate that the receptor may subserve different functions in these two cell populations. (Endocrinology129: 2767–2773, 1991)