We studied the cellular location of HLA-DR alpha-chain synthesis within the human thyroid gland using the technique of in situ hybridization. Tritium-labeled cRNA probes for the HLA-DR alpha-chain DNA sequence revealed significant HLA-DR alpha-chain gene expression in thyroid glands from patients with Graves' hyperthyroidism and Hashimoto's thyroiditis. Hybridization signals, which were RNA strand specific, were widely distributed over thyroid follicular epithelial cells as well as over areas of lymphoid infiltration, with the highest concentration of HLA-DR alpha-chain mRNA within epithelial cells in Graves' disease follicles adjacent to areas of lymphoid infiltration. Qualitatively similar results were obtained with immunoperoxidase staining of thyroid sections for DR antigen. Thyroid tissue obtained from patients who did not have autoimmune thyroid disease contained no detectable HLA-DR antigen, but in situ hybridization revealed variable levels of HLA-DR alpha-chain mRNA in thyroid epithelial cells in such glands. Some glands had no detectable HLA-DR alpha-chain mRNA levels above background, whereas other adult glands had significant DR-specific mRNA levels. Fetal thyroid tissue, however, was negative for strand-specific HLA-DR alpha-chain transcripts as well as for HLA-DR antigen. These results indicate that thyroid epithelial cells are capable of synthesizing HLA class II antigens. The DR genes were expressed to the highest degree within the thyroid glands of patients with autoimmune thyroid disease, where expression was strongly associated with lymphoid infiltration.