The 3H-labeled nucleosides cytidine, deoxycytidine, and thymidine are rapidly incorporated into DNA via dCTP or dTTP pools. Between 30 and 60 min after addition of tracer amounts of a labeled nucleoside to the medium of rapidly growing 3T6 cells, dNTP pools attained a constant specific activity resulting from a steady-state equilibrium between incorporation of nucleoside, de novo synthesis, and linear incorporation of isotope into DNA. Removal of labeled deoxycytidine or thymidine depleted the dNTP pools of isotope within a few minutes and incorporation into DNA stopped. When de novo synthesis of dTTP was blocked with amethopterin, the intracellular dTTP pool rapidly reached the specific activity of thymidine of the medium and isotope incorporation into DNA then measured absolute rates of DNA synthesis. In experiments with and without amethopterin, we found no kinetic evidence for the existence of more than one dTTP pool and the decay of the pool suggested that all dTTP served as precursor of DNA. In contrast, experiments with deoxycytidine and cytidine suggested the presence of separate dCTP pools with preferential DNA synthesis from the pool labeled from cytidine.