To elucidate the biochemical basis of megaloblastic hematopoiesis, the cellular content and metabolism of deoxyribonucleoside triphosphates (dNTPs) were investigated using the bone marrow cells from nine patients with untreated vitamin B12 deficiency and one with folic acid deficiency. The marked imbalance among four dNTPs was noted in all patients. dTTP was invariably elevated rather than depressed. The most striking abnormality, however, was the excessive accumulation of dCTP, which represented the consistent feature exclusive for megaloblastic anemia. Purine nucleotides were also involved to a lesser extent. The apparent turnover pattern of the dTTP pool of megaloblastic anemia marrow cells, in the presence or absence of hydroxyurea, did not differ significantly from that of normoblastic hematopoiesis. The megaloblastic cells assimilated exogenous thymidine into dTTP pool in vitro with enhanced efficiency. It was suggested that the excessive accumulation of dCTP may be related more closely to the pathogenesis of megaloblastic hematopoiesis than to the presumed but not proved deficiency of dTTP.