We have been interested in the fate of nucleotides utilized from the medium by growing or virus-infected Escherichia coli. Our studies of the virus-induced acquisition of deoxycytidine 5'-phosphate hydroxymethylase and of thymidylate synthetase (1-3) and the separation of induction of the two enzymes (4) has led to the possibility of the existence in viral deoxyribonucleic acid of separable polynucleotide sequences. Conceivably a single specific deoxyribonucleic acid fragment might be isolated and applied exogenously to E. coli to induce the formation of a single enzyme under appropriate conditions. In addition, in studies of the mechanism of inhibition of bacterial growth and multiplication by the antitumor agent, 5-fluorouracil, and its derivatives, we demonstrated that a lethal thymine deficiency can be provoked in bacteria (5, 6). The thymine deficiency arises from the inhibition of thymidylate synthetase by the deoxynucleotide, 5-fluorodeoxyuridylate (7). In these experiments the 5-fluorouracil deoxyribonucleoside added to the growth medium was converted to the corresponding nucleotide in the test organism, E. coli. Since the actual inhibitor is a nucleotide in bacteria and apparently in tumor cells as well (8), it was of interest to know whether exogenously supplied nucleotides could penetrate into intact cells. For many years it has been thought that nucleotides and other phosphorylated compounds do not enter cells. Obvious exceptions to this generalization are the incorporation of transforming DNA into bacteria and the existence of nutritional requirements for phosphorylated compounds in some organisms. It has been shown that E. coli actively dephosphorylates exogenously supplied nucleotides (9). However these earlier studies on the possible incorporation of nucleotides labeled with P³² were carried out under conditions now known to be unfavorable (9). The experiments had been designed to minimize dilution of P³² released by a phosphatase, by keeping inorganic P³¹ at a very low concentration in the medium. Since it is now clear that the absence of inorganic P in media containing organic P provokes the development of a powerful phosphatase in E. coli (10), it was possible that E. coli grown under conditions in which the phosphatase was not induced could incorporate intact nucleotides. In approaching this problem we have used a uracil-requiring bacterium in competition experiments, feeding uracil-2-C¹⁴ and unlabeled nucleotide in an inorganic medium containing large amounts of inorganic phosphate. Glucose was the sole source of carbon for compounds other than pyrimidines. It had previously been demonstrated that the organism is completely blocked in the biosynthesis of uracil and that in such competition experiments the pyrimidine of fed deoxycytidine equilibrates completely with fed uracil-2-C¹⁴ in the synthesis of nucleic acid (11). In the present experiments it was observed that fed deoxycytidylate did not so equilibrate with fed uracil. The deoxycytidylate was selectively used for thymidylate synthesis (12). We have explored the nature of this unusual pattern of utilization of the nucleotide compared to the nucleoside in some detail. In addition we undertook to see if a nucleotide of diminished charge such as the methyl ester of deoxycytidylate could penetrate bacteria in a variety of experimental conditions.