The T7 polymerase transcription system was used for in vitro synthesis of unmodified versions of the E. coli tRNA mutants that insert asparagine, cysteine, glycine, histidine, and serine. These tRNAs were used to qualitatively explore the role of some anticodon bases and the discriminator nucleotide in the recognition of tRNA by aminoacyl-tRNA synthetases. Coupled with data from earlier studies, these new results essentially complete a survey of all E. coli tRNAs with respect to the involvement of anticodon bases and the discriminator nucleotide in tRNA recognition. It is found that in the vast majority of tRNAs both of these elements are significant components of tRNA identity. This is not universally true, however. Anticodon sequences are unimportant in tRNA(Ser), tRNA(Leu), and tRNA(Ala) while the discriminator base is inconsequential in tRNA(Ser) and tRNA(Thr). The significance of these results for origin-of-life studies is discussed.