Previous studies suggest that the mechanism of action of the ribosome in translation involves crucial transfer RNA (tRNA)-ribosomal RNA (rRNA) interactions. Here, a selection scheme was developed to identify bases in 16S rRNA that are essential for tRNA binding to the P site of the small (30S) ribosomal subunit. Modification of the N-1 and N-2 positions of 2-methylguanine 966 and of the N-7 position of guanine 1401 interfered with messenger RNA (mRNA)-dependent binding of tRNA to the P site. Modification of the same positions as well as of the N-1 and N-2 positions of guanine 926 interfered with mRNA-independent binding of tRNA at high magnesium ion concentration. These results suggest that these three bases are involved in intermolecular contacts between ribosomes and tRNA.