The recycling of eukaryotic initiation factor eIF-2 requires the exchange of GDP for GTP, in a reaction catalyzed by the reversing factor (RF). Recent studies have suggested that a 60 S ribosomal subunit-bound eIF-2 . GDP complex is an intermediate in protein chain initiation. We have monitored the distribution of RF in heme-deficient and dsRNA-inhibited lysates by immunoblot analysis of sucrose gradient fractions and have compared the distribution with that of eIF-2(α- 3 2 P). RF and eIF-2(αP) were both found to be tightly associated with 60 S and 80 S ribosomes, as their distribution did not change in gradients containing up to 0.1 M K + . The association of eIF-2(α- 3 2 P) and RF with 60 S and 80 S ribosomes was enhanced in the presence of F - , indicating the presence of an endogenous ribosome-associated phosphatase activity which is capable of dephosphorylating eIF-2(αP) in the absence of F - . These observations are consistent with the hypothesis that under physiologic conditions, RF interacts with the 60 S-bound eIF-2 . GDP complex to promote the dissociation of GDP from eIF-2 and the release of eIF-2 from the 60 S subunit as a complex with RF.