Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis. Display omitted •Peptidyl-tRNA stuck inside the bacterial large ribosomal subunit is released by Pth•Pth action requires the retraction of peptidyl-tRNA from the ribosomal catalytic center•Nascent polypeptide folding may prevent peptidyl-tRNA retraction and release by Pth•RQC machinery-mediated alanine tailing facilitates peptidyl-tRNA release by Pth Svetlov et al. show that, following ribosome splitting, peptidyl-tRNA that remains stuck inside the large ribosomal subunit is released by peptidyl-tRNA hydrolase (Pth). This release requires the retraction of peptidyl-tRNA from the subunit to expose its ester bond to Pth. The release is promoted by the C-terminal oligoalanine tailing of the nascent polypeptide.