In heme-deficient reticulocyte lysates, the alpha-subunit of eukaryotic initiation factor-2 (eIF-2alpha) is phosphorylated due to the activation of the heme-regulated eIF-2alpha kinase (HRI). Phosphorylation of eIF-2alpha impairs the guanine nucleotide exchange activity of eIF-2B and thereby inhibits or shuts off protein synthesis. Delayed addition of hemin to shut-off lysates inhibits the eIF-2alpha kinase activity of HRI and restores protein synthesis; under those conditions, the endogenous phosphatase of the lysate dephosphorylates phosphorylated eIF-2alpha and restores eIF-2B activity. In this report we present evidence that the restoration of eIF-2B activity is dependent on the concentration of added hemin and is related to HRI activity in lysates. The recovery of eIF-2B activity is not affected by protein synthesis inhibitors such as cycloheximide, pactamycin and puromycin, which do not affect the eIF-2alpha phosphorylation. Also, the functional eIF-2B activity that is available in hemin-supplemented lysates is not affected by phosphatase inhibitors such as okadaic acid and heat-stable inhibitor-2. However, the recovery of eIF-2B activity that is observed by the delayed addition of hemin to inhibited heme-deficient lysates is reduced by inhibitor-2 and high concentrations of okadaic acid. These findings suggest that a type 1 phosphatase is involved in the recovery of eIF-2B activity and protein synthesis upon delayed addition of hemin to heme-deficient lysates.