Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits. The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex. The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon. eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome. Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join. A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process. It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function.