Escherichia coli mutants which secreted thymidine, thymine, uracil, cytosine, and guanine into the culture medium were isolated. The isolation strategy was based on the combination of a sensitive screening method and a mutant-generating system. The screening method made use of a thyA mutant of E. coli. These cells, when spread on the agar surface with the beta-galactosidase indicator X-gal, will grow into blue colonies if a minute amount of thymidine is supplied to them from a nearby secretor colony. A chemostat was used as a mutant-generating system to select for E. coli mutants that were resistant to inhibitors of the pyrimidine biosynthetic pathway. Although many mutants were selected based on their secretion of thymidine, other kinds of nucleosides and nucleobases, such as cytosine, uracil, guanine, and thymine, were also present in larger quantities. This rational selection strategy should be applicable to other species of micro-organisms for the isolation of better producers of nucleosides. The production of nucleosides and nucleobases by fermentation could then become a possibility.