Three consecutive G-C pairs in the anticodon stem are a key discriminatory feature of initiator tRNA and are required for its selection by IF3. Here, we have mutated two 16S rRNA nucleotides, G1338 and A1339, which provide the sole contact to the G-C pairs of tRNA(fMet) bound to the ribosomal P site. We have tested their effects on translational activities in vivo and have affinity-purified mutant 30S subunits for functional analysis in vitro. Our results are consistent with the formation of Type II and I minor interactions, respectively, between G1338 and A1339 and the anticodon stem of tRNA and suggest that these interactions play a role in tRNA(fMet) discrimination by IF3. Moreover, our findings indicate that discrimination also involves recognition of at least one additional feature of the tRNA(fMet) anticodon stem loop.