Although the recognition of stop codons by class 1 release factors (RFs) on the ribosome takes place with extremely high fidelity, the molecular mechanisms behind this remarkable process are poorly understood. Here we performed structural probing experiments with Fe(II)-derivatized RFs to compare the conformations of cognate and near-cognate ribosome termination complexes. The structural differences that we document provide an unprecedented view of how authentic stop-codon recognition is signaled to the large subunit of the ribosome. These events initiate with very close interactions between RF and the small-subunit decoding center, lead to increased interactions between the switch loop of the RF and specific regions of the subunit interface and end in the adoption of the precise orientation of the RF for maximal catalytic activity in the large-subunit peptidyl transferase center.