During translation, initiation factor 3 (IF3) binds to the small (30S) ribosomal subunit and regulates the fidelity with which the initiator tRNA and mRNA start codon substrates are selected into the 30S initiation complex (30S IC). The molecular mechanism through which IF3 promotes the recognition and signaling of correct substrate selection, however, remains poorly defined. Using single-molecule fluorescence resonance energy transfer, we show that 30S IC–bound Escherichia coli IF3 exists in a dynamic equilibrium between at least three conformations. We found that recognition of a proper anticodon-codon interaction between initiator tRNA and the start codon within a completely assembled 30S IC selectively shifts this equilibrium toward a single conformation of IF3. Our results strongly support a conformational selection model in which the conformation of IF3 that is selectively stabilized within a completely and correctly assembled 30S IC facilitates further progress along the initiation pathway.