Escherichia coli grew exponentially at a reduced rate in the presence of 50 or 100 μg of trimethoprim/ml if the low-molecular-weight products of folate metabolism or their precursors (thymidine, purines, methionine, glycine, and pantothenate) were supplied in the medium. Folate metabolism was inhibited 99.9% by these concentrations of trimethoprim, but a low level of formylation of methionyl transfer ribonucleic acid (met-tRNAF) could be detected. However, in a medium containing all major amino acids, nucleosides, and vitamins, formylation of met-tRNAF was undetectable in the presence of trimethoprim. No other amino-masked amino acids were detected, and methionine remained a major amino-terminal amino acid of mature proteins. met-tRNAF was rapidly labeled with exogenous methionine and was associated with 30s ribosomal subunits and 70s ribosomes. It was concluded that initiation of protein synthesis can occur with unformylated met-tRNAF in E. coli. Changes in macromolecular composition were associated with the lack of formylation, in particular a fourfold increase in both met-tRNAF and ribosomal subunits. These changes would tend to compensate for the low specific rate of initiation with unformylated met-tRNAF.