T4 type II 5'-deiodinase (5'-D II) activity was studied in wild-captured Richardson's ground squirrels. As previously reported for other species, 5'-D II activity was detected in frontal cortex, cerebellum, pineal gland, and brown adipose tissue (BAT); in the median eminence the levels of 5'-D II activity were undetectable with our methodology. When pineal gland, frontal cortex, and cerebellum nyctohemeral profiles were studied, none of them showed variations. Cold exposure for 4 h led to an increase in the enzymatic activity 10-fold above the basal values for BAT, while in the pineal gland the values were doubled; cold exposure failed to change the 5'-D II activity in the frontal cortex. Acute melatonin treatment caused a 7-fold increase in 5'-D II activity in BAT, but did not affect enzyme activity in either the pineal gland or frontal cortex. The data indicate that 5'-D II in Richardson's ground squirrel shows classical localizations. Additionally, two new regulatory factors of 5'-D II are reported, i.e. melatonin for BAT and cold for the pineal gland.