Immunoreactive β-endorphin (ir-βEP) and immunoreactive N-acetyl-endorphin (ir-NacEP) have been demonstrated in the rat thyroid by specific RIA and characterized by reverse phase HPLC. In addition, pituitary and thyroid ir-βEP and ir-NacEP levels have been determined after manipulation of the pituitary-thyroid axis by chronic (21 days) treatment with 1) propylthiouracil (PTU), 2) L-T4, 3) L-T3, or 4) T3 plus PTU. No difference in anterior pituitary or neurointermediate lobe ir-βEP was seen between controls and treated groups (n = 8/group). In contrast, levels of ir-NacEP were markedly lower (P < 0.01) in both hypo- and hyperthyroid groups than in controls, in both anterior pituitary and neurointermediate lobe. In the thyroid, levels of both ir-βEP and ir-NacEP were profoundly depressed (P < 0.01) in all treated groups, with no change in calcitonin levels, suggesting that the thyroid effect of PTU and T3/T4 may be specific for the synthesis, processing, and/or release of pro-opiomelanocortin derived peptides. The findings in this study suggest 1) that acetylation of pituitary and thyroid βEP is similarly sensitive to PTU and thyroid hormone administration and 2) that in the thyroid, but not in the pituitary, both PTU and thyroid hormones markedly lower levels of pro-opiomelanocortin-derived peptides.