The activities of thymidylate synthetase and thymidine kinase were compared in tissues of normal (adult and developing), cortisol-injected, and tumor-bearing rats. The purpose of the study was to determine whether the activities of these two enzymes, which catalyze reactions leading to the same metabolic intermediate, changed proportionately, reciprocally, or independently under different physiological conditions. Both enzymes had high activities in fetal tissues. Thymidine kinase concentrations decreased shortly before or immediately after birth; in several tissues, transient postnatal peaks in thymidine kinase activities appeared within the first 3 weeks after birth. Thymidylate synthetase activities declined gradually after parturition and showed no significant postnatal rises. In sucklings given injections of cortisol, thymidine kinase activities were reduced substantially in eight tissues while thymidlyate synthetase decreased only in lung and thymus of 11-day-old rats. In tumor-bearing rats, thymidine kinase activity increased dramatically in spleen, whereas thymidylate synthetase activities only doubled. In host liver, rises in thymidine kinase activities were not always matched by increases in thymidlyate synthetase. In the tumors, both activities were higher than in most normal adult tissues. Despite the differential sensitivities of the two enzymes to cortisol and tumor bearing, thymidylate synthetase and thymidine kinase were closely correlated in tissues of untreated animals. The Spearman rank correlation coefficient for 112 tissues was 0.895, while the correlation coefficient between the standard scores of the activities was 0.839. The activities of the two enzymes did not appear to be reciprocal or compensatory during normal differentiation or during dedifferentiation associated with tumor bearing, but their potentials for activity were independent of each other.