In female mice (30-35 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., there was a 24-hr cycle change (P < 0.0001, Cosinor analysis) in the activity of hepatic orotate phosphoribosyltransferase (OPRTase; EC 22.214.171.124), uridine phosphorylase (UrdPase; EC 126.96.36.199), and dihydrouracil dehydrogenase (DHUDase; 188.8.131.52) but not thymidine phosphorylase (EC 184.108.40.206). The peaks of both OPRTase and UrdPase activities occurred in the activity span at around 18 and 15 hours after light onset (HALO) and the trough at 6 and 3 HALO, respectively, when samples were taken every 4 hr. Conversely, the peak of DHUDase occurred in the rest span at around 3 HALO and the trough at 15 HALO. The maximal enzyme activities (3146 ± 172, 561 ± 25, and 6.7 ± 0.7 pmol/min/mg protein) was 210, 400 and 560% higher than the minimal activities (1507 ± 172, 139 ± 25, and 1.2 ± 0.7 pmol/min/mg protein), for OPRTase UrdPase, and DHUDase, respectively. A circadian rhythm 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 UrdPase and DHUDase activities with a period of 1 hr difference in the time of maximum and minimum enzyme activities. OPRTase, on the other hand, showed little change after 4 weeks of adaptation under the reverse light cycle. The circadian rhythm of these key enzymes of pyrimidine metabolism, the interrelationship of their activities, and their role in the regulation of uridine bioavailability could be of particular significance in modulating the therapeutic regimens with 5-fluorinated pyrimidines. © 1993.