Thymidylate (dTMP) synthase (EC 18.104.22.168) activity was measured in 100,000 x g supernatant fluid with a sensitive, rapid radio assay. The activity in normal rat liver was low (0.098-0.204 nmol/hr/mg protein). dTMP synthase specific activities in rat thymus, spleen, bone marrow, testis, lung, heart, brain, kidney, and small intestine were 6297, 1842, 1500, 788, 215, 76, 61, 39 and 24%, respectively, of that of the liver. The activity in 5-day-old rat liver was 16-fold higher than in adult. dTMP synthase activity increased in rat hepatomas to 7- to 125-fold of that of normal rat liver. There was a significant correlation between the increase in synthase activity and the proliferation rates of the hepatomas. In 8 human colon carcinomas, dTMP synthase activity increased to 2.9- to 8-fold of that of normal human colon mucosa. In leukemic leukocytes from 3 leukemia patients, activity was 8- to 10-fold higher than in normal leukocytes.