The activity of thymidine kinase (TK, EC 2.7.1.21), the first enzyme of the thymidine phosphorylation pathway, was measured at various times over a 24-hr period in the spleens of Sprague-Dawley rats that had been housed under standardized conditions of light and dark for at least 4 weeks before the study. Spleen cytoplasmic TK activity was assayed with [2-14C]thymidine as substrate. Under "normal" light conditions (lights on 6:00 a.m.-6:00 p.m. and lights off 6:00 p.m.-6:00 a.m.), a circadian variation of TK activity was observed (P < 0.0001), Cosinor analysis) with peak activity (1.98 nmol product/hr/mg protein) at 1:00 a.m. (19 hr after light onset, HALO) and trough activity (0.40 nmol product/hr/mg protein) at 1:00 p.m. (7 HALO). Maximum enzyme activity exceeded minimum activity by approximately 5-fold. Reversing the light-dark cycle resulted in a corresponding shift in TK activity. Under these "reverse" conditions (lights on 6:00 p.m.-6:00 a.m. and lights off 6:00 a.m.-6:00 p.m.), a circadian variation in TK activity was also observed (P < 0.0001, Cosinor analysis) with peak activity (1.14 nmol product/hr/mg protein) at 12:00 noon (18 HALO) and trough activity (0.32 nmol/hr/mg protein) at 12:00 a.m. (6 HALO). Maximum enzyme activity exceeded minimum activity by approximately 4-fold. In summary, this study demonstrated for the first time that TK activity varies over a 24-hr period in association with the light-dark cycle.