Isolates with mutations in glyS, the structural gene for glycyl-transfer ribonucleic acid (tRNA) synthetase (GRS) in Escherichia coli, are frequently found among glycine auxotrophs. Extracts of glyS mutants have altered GRS activities. The mutants grow with normal growth rates in minimal media when high levels of glycine are provided. No other metabolite of a variety tested is capable of restoring normal growth. The glyS mutants fail to make ribonucleic acid (RNA) when depleted of exogenous glycine in strains which are RC(str) but do so when the cells are RC(rel). In contrast, biosynthetic mutants which are unable to synthesize glycine (glyA mutants) do not make RNA when deprived of glycine even if they are RC(rel); in this case, RNA is synthesized upon glycine deprivation only when the nucleic acid precursors made from glycine are provided in the medium. The level of serine transhydroxymethylase is unaltered in extracts of any of the glyS mutants, even though the level of charged tRNA(Gly) is at least 20-fold lower than that found in a prototrophic parent; this indicates that, if there is control over the synthesis of serine transhydroxymethylase, it is not modified by reduced levels of charging of the major species of tRNA(Gly).