The nucleotide sequence of an E. coli isoleucine tRNA (tRNAIle minor) specific for the codon AUA was determined by postlabeling procedures using only 2.5 micrograms (0.05 A260 unit) of the material. The sequence was pG-G-C-C-C-C-U-s4U-A-G-C-U-C-A-G-U-Gm-G-D-D-A-G-A-G-C-A-A-G-C-G-A-C-U-N+-A-U-t6A-A-psi-C-G-C-U-U-G-m7G-acp3U-C-G-C-U-G-G-T-psi-C-A-A-G-U-C-C-A-G-C-A-G-G-G-G-C-C-A-C-C-AOH. The nucleotide sequences in the regions of the D arm and T psi C arm of tRNAIle minor were quite similar to the corresponding regions of tRNAIle major. However, the sequences in the CCA stem and anticodon stem of tRNAIle minor were different from those of tRNAIle major. The overall homology between the two isoleucine tRNAs was 68%. E. coli tRNALys, tRNAMet, tRNAValIIA and tRNAArg also have relatively high sequence homology with tRNAIle minor.