Twenty-four hour rhythms of histamine (H), its biosynthetic enzyme, L-histidine decarboxylase (EC 4.1.1.22, HD) and its inactivation enzyme, histamine N-methyltransferase (EC 2.1.1.8, HMT) were assayed in the 63-day-old male rat hypothalamus using a modified single isotopeenzyme microassay. H was found to have a 24-h rhythm with a maximum concentration at 11:00 h (lights on 10:00-22:00 h) and a minimum at 23:00 h. In vitro HD and HMT activities were also found to have 24-h rhythms with peak activites in both occurring at 04:00 h. HMT also had a broad secondary peak throughout the day, whereas HD activity during the day was dominated by a mid-light trough at 16:00 h. These rhythms are considered significant for evaluation of the control and physiological role of histamine in the mammalian hypothalamus.