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A eukaryotic initiation factor 2 (eIF-2)-ancillary protein factor Co-eIF-2 promotes displacement of GDP from eIF-2GDP and facilitates ternary complex (Met-tRNAf• eIF-2-GTP) formation in the presence of Mg2+. Heme-regulated protein synthesis inhibitor, HRI, phosphorylates the α-subunit of eIF-2 and thus inhibits ternary complex formation as Co-eIF•2 does not displace GDP from eIF-2α(P)•GDP. RF, a high molecular weight cell supernatant factor, reverses protein synthesis inhibition in heme-deficient reticulocyte lysates and also reverses HRI inhibition of ternary complex formation. RF contains Co-eIF•2 activity. In addition, an active RF preparation contains excess α-subunit of eIF-2 in the free and unphosphorylated form and this α-subunit of eIF-2 is not phosphorylated by HRI and ATP. In this paper we report (i) an active RF preparation contains excess α-subunit of eIF-2 and this α-subunit can be phosphorylated by HRI and ATP in the presence of GDP; (ii) RF promotes ternary complex formation by elF- 2•[3H]GDP with accompanying GDP displacement; (iii) in the presence of HRI and ATP, RF promotes ternary complex formation by eIF-2•[3H]GDP without accompanying GDP displacement; (iv) in the presence of HRI and ATP, the ternary complex formed using RF is active in Met-tRNAf 40S initiation complex formation; (v) both the ternary complex and the Met-tRNAf 40S complex formation in the presence of HRI and ATP are completely inhibited by prior incubation of RF with GDP; (vi) upon further fractionation of an active RF fraction, a preparation can be obtained that contains HRI-sensitive CoeIF- 2 activity. However, this preparation does not efficiently reverse protein synthesis inhibition in heme-deficient reticulocyte lysates and does not contain excess a-subunit of eIF-2. Based on these observations, we have suggested (α) RF provides the unphosphorylated a-subunit to eIF-2a(P)-GDP and restores eIF-2 activity. This RF activity is inhibited as the α-subunit in the RF preparation becomes phosphorylated by HRI and ATP in the presence of GDP; (b) RF contains Co-eIF- 2 activity, which has dual functions: (i) stimulation of ternary complex formation by eIF-2 and (i) GDP displacement from eIF-2•GDP during ternary complex formation. In the presence of HRI and ATP, Co-eIF-2 still stimulates ternary complex formation by unphosphorylated eIF-2 but does not displace GDP from eIF-2α(P)•GDP.