A commonly used strain of Escherichia coli K-12 was shown to be deficient in the transport of a number of nucleosides, including thymidine. Thymidine incorporation was unaffected. Strain AB2497 exhibited a strikingly lower thymidine pulse-label incorporation at low (less than 1 mug/ml) thymidine concentrations than do many other strains. The deficiency appeared to be due to mutation in a single gene. This gene, which we designated nup (for nucleoside uptake), is located at 10 to 13 min on the E. coli linkage map. In nup+ strains, the transport of a given nucleoside was relatively insensitive to large excesses of other nucleosides but was competitively inhibited by the same nucleoside. Mutants deficient inthymidine kinase are deficient in thymidine uptake but normal in deoxyadenosine uptake. A two-step model for nucleoside transport is presented in which the first step, utilizing the nup gene product, is a nonspecific translocation of nucleoside to the interior of the cell. In the second step, the individual nucleosides are modified by cellular enzymes (e.g., nucleosides kinases) facilitate accumulation.