The response of type-II thyroxine 5′-deiodinase (5′-DII)DII) to melatonin treatment was studied in the Syrian hamster. Male hamsters were treated for 15 days with a s.c. pellet containing melatonin, and 5′-DII activity in brown adipose tissue, anterior pituitary gland, Harderian gland and pineal gland was measured using a radioenzymatic technique. Melatonin-treated animals exhibited enhanced 5′-DII activity restricted to brown adipose tissue; the increase was threefold above the values measured in the control group. Serum concentrations of thyroid hormones were unaffected by melatonin treatment. We conclude that the stimulatory effect of melatonin on type-II thyroxine 5′-deiodination is specifically directed to the isoenzyme located in brown adipose tissue and is not accompanied by changes in serum thyroid hormones.