The elucidation of the role of release factor 3 (RF3) in translation termination and the demonstration that GTP hydrolysis was needed for RF3 action became possible with the development of improved purified in vitro systems for protein synthesis. In the absence of RF3, the inhibitory action of RF1 on ribosome recycling can be reduced by increasing the ribosome-recycling factor (RRF) concentration, suggesting that whatever the action of RRF may be, it is incompatible with the presence of RF1 on the ribosome. The experiments in vitro with the purified translation system show that ribosome recycling from initiation via protein elongation and termination of protein synthesis depends strictly on the presence of IF3. This implies that RRF and EF-G together split the ribosome into its subunits after termination and that this step is the overture to ribosome recycling back to initiation of translation from the posttermination state. The fate of the mRNA after termination and ribosome recycling is highly relevant for translation of multicistronic mRNAs. The in vitro data suggest that when termination of protein synthesis is followed by the action of RRF, EF-G, and IF3 the 30S particle remains attached to the mRNA, allowing the latter to diffuse long distances along the subunit. In the presence of GTP, RF3 accelerates recycling of RF1 and RF2 by enhancing their rates of dissociation after termination of translation.