The activity of hepatic uridine phosphorylase (EC 2.4.2.3.) in male mice (24-29 g) maintained in standardized conditions of 12 hr light (0600-1800 hr) alternating with 12 hr darkness (1800-0600 hr), food and water ad lib., exhibited a circadian rhythm (P less than 0.0001, Cosinor analysis). The peak of enzyme activity (559 +/- 25 pmol/min/mg protein) occurred at 15 hr after light onset (HALO) with the nadir (139 +/- 25 pmol/min/mg protein) at 3 HALO when samples were taken every 4 hr. Female mice showed essentially the same pattern. A circadian rhythm (P less than 0.0001, Cosinor analysis) was also observed when the light-dark cycle was shifted (reverse cycle) so that the lights went on at 2200 hr and off at 1000 hr. Under the reverse cycle condition, there was a corresponding shift in the enzyme activity with a lag period of 3.5 hr in the time of maximum and minimum enzyme activities (i.e. the peak at 11 HALO and the nadir at 23 HALO) after a 2-week adaptation period. The lag period was reduced to 1 hr after 4 weeks of adaptation, and no further change was observed after 6 weeks of adaptation. The plasma concentration of uridine also exhibited a circadian rhythm (P less than 0.0001, Cosinor analysis) with peak concentration (10 microM) occurring at 2 HALO and a nadir (5 microM) at 14 HALO. The circadian rhythm observed in the plasma concentration of uridine is the inverse of that for uridine phosphorylase activity. These results demonstrate that hepatic uridine phosphorylase plays an important role in the regulation of the uridine level in the blood which, in turn, may be involved in the humoral control of sleep by uridine. This may also be of clinical significance in enhancing the antitumor efficacy of the 5-fluorinated pyrimidines by modulating the time of their administration.